It's no secret that Mario has a huge assortment of Mario characters. This not only include any playable characters that the series introduced, but also the non-playable characters and enemies are also part of Mario's huge cast. However! If you have taken a glance at the Mario games, you might notice that there are characters introduced in the games that are practically one-off or rarely-recurring, where they appear in only one game and never again. As an example, in Mario Golf (N64), five human characters (Plum, Maple, Sonny, Charlie and Harry) were introduced but they never appeared again. Enemies are not immune to this, such as Hot Foot from Super Mario Bros. 3. Very few games have every character/enemy reappear in future games, with the first Super Mario Bros. being the most prominent example.
With that said, there are quite a number of Mario characters that are being used regularly since being introduced, some of them having a short break. Besides Mario himself, we have Luigi, Peach, Toad, Yoshi and Bowser as the most famous examples, and on a smaller scale, we even have Birdo, Daisy, Wario, Waluigi, Donkey Kong, Diddy Kong, Bowser Jr. and Toadette. Some people may argue about the status of Daisy and Waluigi as major characters, but for the definition of this topic, they count because they made regular appearances even though it's outside the Super Mario series, which is something even characters introduced in a Super Mario game can't brag, like Il Piantissimo or even Lubba. Not to mention, Daisy and Waluigi have regular appearances in group shots. Enemies can even make prominent appearances too, for we have Koopa Troopa, Goomba and Boo as major recurring enemies.
So far, you might notice that I didn't mention Rosalina, and the reason is this: Rosalina is the last character in recent memory that has regular appearances, and thus counted as a major Mario character. Every other character I have mentioned were introduced before Rosalina, who herself first appeared in Super Mario Galaxy, a 2007 game. I feel that Rosalina wasn't really made out to be a major character early on, based on the fact that she has a backstory in the form of a storybook and had more than 2 names in different languages, which to me, indicates that they only planned for Rosalina to appear in this game until someone decided that Rosalina is worth being recurring. Earlier on, Rosalina wasn't quite prominent in her appearances, with the next big one being Mario Kart Wii, but starting in 2013, her status as a major Mario character is unquestioned, when there was quite a combo of Super Mario 3D World, Mario Kart 8, Super Smash Bros. 4, and even Mario Party 10 that put her in the spotlight. After Rosalina though, we might not even have a next major Mario character. Captain Toad was introduced in the same game as Rosalina, so he technically doesn't count.
On the realm of spin-offs, Mario Kart is the biggest contender in introducing major characters, since they did introduce Toadette, after all. The biggest recent memory is Pink Gold Peach, who was such a surprise hit but probably won't stick as a character, since she is after all, a derivative of an existing character. After that one, Baby Daisy and Baby Rosalina are more or less low-priority recurring characters that will probably only be used in Kart games from this point. Outside Mario Kart, the only character that managed to slip into the ranks of the major recurring characters is of course Waluigi. Even though many people shunned him for gaining prominence outside the main or Kart games, I am fascinated by what many consider an "accident" since he is quite an enigmatic and fun character that other developers and Martinet can have fun with, and because he keeps appearing in different spin-off games (over the majority of those from Super Mario ones), I feel there is something the developers like about him too.
One issue I can see is, based on an offhand post I read regarding characters, Nintendo has a quality assurance team for characters, including positions for Character Supervisor (check Mario Kart 8 Deluxe and Mario Tennis Aces' credits), which is probably due to Nintendo regarding their characters and worlds as valuable that they stopped the developers from doing experimentations, likely during the DS/Wii era (It's probable that this form of management is the reason that Paper Mario didn't have a world of original characters, based on my understanding of this situation).
Even in the Super Mario games, there aren't too many new additions and some of them probably aren't staying around too much. Pom Pom made around a few appearances after 3D Land, Nabbit was introduced in New Super Mario Bros. U and was made playable in New Super Luigi U, but really only made like three appearances outside that game. Sprixie Princess is not that prominent as she only appears in one other game. Mary O. is probably just stuck in Super Mario Maker due to the different style of game. And then, the Broodals is probably never going to stick around after their only appearance in Super Mario Odyssey. It remains to be seen if Cappy will stick around, or if he gets to reach new places outside of the game he was introduced in.
So, are there any characters that could be considered major, after Rosalina?
Thank you for reading.