Abstract It was carried out a cytogenetic comparison between Phyllostomus hastatus and Artibeus lituratus (Chiroptera, Phyllostomidae), using G-, C- and NOR banding techniques. The P. hastatus specimens presented 2n=32 chromosomes, including 13 biarmed autosome pairs and one acrocentric. The X chromosome is submetacentric and the Y is a minute acrocentric. A. lituratus has 2n=30 chromosomes in females and 31 in males specimens. The autosomes are 14 biarmed pairs and the chromosomal sexual system is XY1Y2 type, being the X chromosome a large submetacentric and both Y1 (real Y) and Y2 acrocentric elements. Several chromosomal homologies are shared between both taxa, including whole chromosomes and chromosomal arms. Based on similarity of G-banding pattern is proposed that the system XY1Y2 from A. lituratus risen by a tandem fusion event involving the stenodermatine original biarmed X chromosome with the autosome acrocentric homologous to Y2.