To study European bat lyssavirus (EBLV) in bat reservoirs in the Netherlands, native bats have been tested for rabies since 1984. For all collected bats, data including species, age, sex, and date and location found were recorded. A total of 1,219 serotine bats, Eptesicus serotinus, were tested, and 251 (21%) were positive for lyssavirus antigen. Five (4%) of 129 specimens from the pond bat, Myotis dasycneme, were positive. Recently detected EBLV RNA segments encoding the nucleoprotein were sequenced and analyzed phylogenetically (45 specimens). All recent serotine bat specimens clustered with genotype 5 (EBLV1) sequences, and homologies within subgenotypes EBLV1a and EBLV1b were 99.0%–100% and 99.2%–100%, respectively. Our findings indicate that EBLVs of genotype 5 are endemic in the serotine bat in the Netherlands. Since EBLVs can cause fatal infections in humans, all serotine and pond bats involved in contact incidents should be tested to determine whether the victim was exposed to EBLVs.