A 15-year cytogenetic survey on one population of the leaf litter frog <i>Eleutherodactylus maussi</i> in northern Venezuela confirmed the existence of multiple XXAA♂/XAA^Y♀ sex chromosomes which originated by a centric (Robertsonian) fusion between the original Y chromosome and an autosome. 95% of the male individuals in this population are carriers of this Y-autosome fusion. In male meiosis the XAA^Y sex chromosomes pair in the expected trivalent configuration. In the same population, 5% of the male animals still possess the original, free XY sex chromosomes. In a second population of <i>E. maussi</i> analyzed, all male specimens are characterized by these ancestral XY chromosomes which form normal bivalents in meiosis. <i>E. maussi</i> apparently represents the first vertebrate species discovered in which a derived Y-autosome fusion still coexists with the ancestral free XY sex chromosomes. The free XY sex chromosomes, as well as the multiple XA^Y sex chromosomes are still in a very primitive (homomorphic) stage of differentiation. With no banding technique applied it is possible to distinguish the Y from the X. DNA flow cytometric measurements show that the genome of <i>E. maussi</i> is among the largest in the anuran family Leptodactylidae. The present study also supplies further data on differential chromosome banding and fluorescence in situ hybridization experiments in this amphibian species.