Her lost brother is Prince Mush. She is the first to catch on to Grubba's plot to use the Crystal Stars to make himself eternally youthful by sapping other fighter's powers. She then tips Mario under the name 'X', sending him e-mails that include clues to find out about Grubba's role in the missing fighters.
After the fight with Grubba, she appears and demands Grubba tell her where her brother is, after a discussion with Grubba, her brother, Prince Mush, appears out of Grubba's device and the two are reunited. She then gives Mario the Crystal Star and assumes the position of the Glitz Pit manager in place of the defeated Grubba.
KP Pete tells Mario that he has a crush on "The Cutie at the Watering Hole," who appears to be Jolene. Similarly, Bandy Andy is also implied to have a crush on Jolene, often stalking her.
Jolene helping Mario under the alias of "X" could be a reference to the "Racer X", who helped Speed Racer in the Speed Racer Series.
Unlike normal female Toads (including Toadette), Jolene's shoes appear to possess something akin to a high heel. They appear red when in her suit, while they appear black when paired with a black dress as seen in an above image.