Johto was the first Pokémon game region to feature 100 newly introduced species.
The expansion arrived with Pokémon Gold and Silver, the second-generation Game Boy Color games. Alongside the new Johto setting, the games added 100 species numbered 152 through 251 in the National Pokédex. Chikorita, Cyndaquil, and Totodile were among the new Pokémon introduced at the start of the adventure.
Gold and Silver also added the Dark and Steel types, Pokémon breeding, held items, and differently colored Shiny Pokémon. These features made the sequel more than a simple collection of new creatures; they broadened the systems established by the original games.
Kanto is the first region and introduced the original 151 species, so it is not the answer. Hoenn followed Johto in Pokémon Ruby and Sapphire and introduced the next generation of species.