3. Tyler Johnston
Tyler Johnston first made his appearance in the episode Bugs of Season 1 as Matthew Pike, he assists Sam and Dean Winchester. He has a deep interest in insects and science. By the end up of the episode, his relationship with his father is improved. Tyler also appeared as the angel Samandriel in Season 8. He would be captured by Crowley who captured him and hacked his mind by torturing. He believed in Castiel, but tragically he was killed by Castiel under the orders of Naomi.
2. Carrie Anne Flemming
Carrie Anne Flemming has appeared in popular shows such as iZombie and Masters of Horror. In Supernatural, she first appeared as a nurse in the Season 2 Episode ‘In My Time Of Dying’. The episode saw Dean hanging between life and death. Ultimately John makes an ultimate sacrifice to save his son. She would later reappear, but playing a different character of Karen Singer. Karen Singer was possessed by a demon and she tried killing Bobby when she became rabid. She would die of her wounds. She would be resurrected by the Death in order to send a message to Bobby. Sadly, Bobby is forced to kill her again when she begins turning.
1. Alexia Fast
Alexia Fast has also played two different roles in the Supernatural Universe. She first played the role of Missy Bender, the youngest of the Bender Family in Season 1 Episode ‘The Benders’. She is evil and pure psychotic. When Dean mistook her for an innocent girl, he let Missy stab her. Missy is ultimately arrested at the end of the episode. She would later appear in Season 7 Episode ‘The Slice Girls’, she played Emma who is an Amazon conceived when Dean and Lydia had a one-night-stand. She would die at the hands of Sam when she tried killing Dean.
Apart from these, there are several other actors who have also played multiple roles on the show. This includes the actor Ian Tracey who played the role of a hunter named Lee Chambers in S7 who raised his daughter in the lifestyle. He was killed by a vampire on the orders of Victor in order to turn Krissy into a hunter by forcing her to choose the hunter’s lifestyle. Ian would later reappear in Season 12 as the angel named Ishim, the powerful angel under whom Castiel once served. He gets into a fight with Lily and was about to kill her when Castiel stabs him from behind. Lily has been seeking revenge against him for over a hundred years. Also, do let us know if we missed out any other actors who played several different roles in SPN Universe.
Source: Supernatural Wikia