Niche expansion and shifts are involved in the response and adaptation to environmental changes. However, it is unclear how niche breadth evolves and changes toward higher-quality resources. Myotis pilosus is both an insectivore and a piscivore. We examined the dietary composition and seasonality in M. pilosus and the closely related Myotis fimbriatus using next-generation DNA sequencing. We tested whether resource variation or resource partitioning help explain the dietary expansion from insects to fish in M. pilosus. While diet composition and diversity varied significantly between summer and autumn, the proportion of fish-eating individuals did not significantly change between seasons in M. pilosus. Dietary overlap between M. pilosus and M. fimbriatus during the same seasons was much higher than within individual species across seasons. We recorded a larger body size, hind foot length, and body mass in M. pilosus than in M. fimbriatus and other insectivorous trawling bats from China. Similar morphological differences were found between worldwide fishing bats and non-fishing trawling bats. Our results suggest that variation in insect availability or interspecific competition may not play important roles in the dietary expansion from insects to fish in M. pilosus. M. pilosus has morphological advantages that may help it use fish as a diet component. The morphological advantage promoting dietary niche evolution toward higher-quality resources may be more important than variation in the original resource and the effects of interspecific competition.