Well, I'm gonna have to go with the obvious here with Sam dying and Dean making the deal in All Hell Breaks Lose but there are a lot of other tragic moments like John dying, Mary making the deal despite Dean's efforts to stop it but to me Sam dying and the consequences of that were the most tragic.

first thing that comes to mind is the pilot, actually ! mary and jess die, two events that change the course of the lives of these brothers, and that will always haunt them.

it sets the tone for the whole series to commence with tragedy.

Dean dying or Sam doing was pretty tragical but they both came back, Mary and Jessica dying also tragical but didn't "care" much cause I didn't get to know the characters like I got to know Dean & Sam or even John..
The most tragical is at the episode "When the Levee breaks" What happens between the brothers!It was the most painful think to watch that last fight scene,Sam leaving and Dean on the floor!That's tragic for me!

omg youre right !

I still haven't watched this episode again,total 2 times and I can't another one!
I get a feeling in my stomach and sadness feels me up..
Many scenes are tragic but that look Sam have over Dean and Dean's face when Sam leaves,that works for me as ultimate tragic moment!

I agree!Breaks my heart!the scene I can't watch after a while is when Dean talks on the Impala next to Sammy about what happened to hell.That's my tragical scene.

5x10. The most heartbreaking episode for me.

I think Asylum, Dream a little Dream of Me and Dark Side of the Moon are few of the tragic episodes for me. I really felt sad to see Dean craving for his mothers touch in 5x16.

"Swan Song" Every time I see Dean on this picture I could cry again. I don't know why, but looking at him like this, desperate, helpless, and totally lost for what happend to Sam, makes me go back in the time... Dean is running with a baby Sam, Dean is taking care of a little brother Sam, Dean is protecting Sam. And that last scene on the car where Dean is beaten by Sam/Lucifer. The way he was telling him:" It's ok Sammy....it's ok" I really gonna make myself cry again:( And Sam... my poor baby. So lost, but strong enough to stop Lucifer of killing Dean, and the moment when he jumped in that hole... This is exactly how I reacted, like Dean on this picture. Just to mention that this moment of Dean is so strong and powerful, that I decided to keep it in my memory like the best Dean/acting moment I'v seen!

in season 2, when sam died in the arms of his brother and dean brought him back solding his soul to lillith..that was a great tragical moment also when bobby learned that too
but spn is a tragical show so many more scene or episode tragical..for me, this one was the more stronger because after that it was always the same stuff...