The only representatives of Emballonuridae in the Neotropics are Diclidurini from the subfamily Emballonurinae. In this tribe, karyotype comparison based on G-banding patterns has revealed extensive chromosomal divergence. Here, we present for the first time a detailed karyotype analysis of a Diclidurini species, Saccopteryx bilineata (SBI), by G-banding and cross-species fluorescence in-situ hybridization (FISH) with Myotis myotis (Vespertilionidae) painting probes. The 25 evolutionarily conserved units (ECUs) ancestral to all Chiroptera were found as 37 segments on the 2n = 26 SBI karyotype. Comparison with the karyotype of another Emballonuridae (Emballonura monticola) from South-East Asia revealed a single common derived segment combination, MMY3+MMY22i, pointing to a fast karyotype evolution in Emballonurinae.