Throughout the series, Sam and Dean have often been mistaken for a couple, much to their collective confusion.
While investigating a death in the Oasis Plains community, Sam and Dean meet with Larry Pike at a barbecue, who tells them they accept all homeowners regardless of race, religion, color, or sexual orientation. An embarrassed Larry is corrected by Dean that they are in fact brothers.
Inside the barbecue Sam and Dean meet Lynda Bloome, who tells them the same thing Larry did. Dean with a slight chuckle excuses himself, calling a surprised Sam "honey" and smacking him on the rear before leaving.
While getting a room at the Court Motel in Fitchburg, Wisonsin, Dean tells the young clerk Michael he wants two queens. Michael seeing Sam waiting outside snickers and under his breathe says "I'll bet."
Checking in at the Pierpont Inn under the guise of antiquing, Susan Thompson tells Sam and Dean they "look the type" and asks if they want a king size bed. Embarrassed, Sam quickly tells Susan they are just brothers and want two singles, while a confused Dean asked what she meant by that they look the type.
In a twist Dean plays up to the fact that a shopkeeper assumes they are a couple and has Sam ask about a Christmas wreath they saw at a couple's home, prompting Sam to say it was "yummy."
After reuniting with Sam at his motel, Ruby asks if they are "together" which Sam tells her that Dean is his brother.
After learning about the existence of the Supernatural books, Sam and Dean learn that there are fans, some of who ship Sam and Dean together. When Dean asks if they realize they are brothers, Sam tells him it "doesn't seem to matter."
Michael Wheeler and Brian Wilcox secretly film Sam and Dean who are investigating a case as FBI agents. Michael tells Brian he is getting a workplace romance vibe from Sam and Dean and later Brian tells him of their stake out at a diner how they talked about being apart for a year, believing they were right about the romance.
When Dean sees Siobhan and Maggie standing close to each, he asks Marie why what they are doing. She tells them them rehearsing a "brotherly moment" scene, and begins describe how Sam and Dean are bonded, which leads to question why they are so close together. Marie tells him "reasons", which prompts Dean to ask if she realizes they are brothers. With Marie telling him, "Well, duh ! But... Subtext"
- Mistaken for Gay on TV Tropes