If remakes don't count, I'm going to go with my first playthrough of a game.
Gym 1 - Brock: Brock was hard in Yellow because all I had was Pikachu when I first battled him.
Gym 5 - Sabrina: Since psychic types didn't really have much of a weakness in Gen I (you can say Gastly, Haunter, and Gengar, but since they're part poison, throw a psychic at them and they'll faint easily), she was really hard.
Gym 3 - Whitney: Underestimating normal type Pokémon round 1. Miltank was just a powerhouse.
Gym 5 - Norman: Underestimating normal type Pokémon round 2. That Slacking was just as much of a powerhouse as Miltank.
Gym 3 - Maylene: I didn't know Lucario's typing when I first battled it in Diamond, so I lost to it a lot before finally going to look it up.
Gym 2 - Lenora: Underestimating normal type Pokémon round 3. Retaliation and that powerhouse of a Herdier combination is deadly.