You would have to include acting bosses if you want to consider either Crea or Ligambi because that's what they were/are.

Anyway, if the question is longest running since 2000, it would be Vic Amuso and Carmine Persico because they have all been the official boss of their families over that time period. John DiFronzo, also, but he may no longer be boss so I'm not sure about him. I don't recall off the top of my head exactly when Joey Merlino became official boss after Natale flipped.