ABSTRACT Previous studies on linkage disequilibrium involving four tightly linked genes that code for the alpha-esterases of Drosophila montana suggest that these loci arose from a primitive esterase gene by gene duplication, Iollowed by tandem duplication (ROBERTS and BAKER 1973). We have examined the esterase variants in the closely related species, Zacicola, fluvomontana and borealis. These studies reveal that borealis has only a single esterase locus, and flmomontana may have only two loci. Cytological studies, using aceto-orcein staining and Hoechst fluorescence of squashes of ganglion chromosomes, reveal acrocentric Y chromosomes for all six species of the montana phylad, with the exception of borealis, which has the primitive rod-shaped Y chromosome. These studies provide evidence ayainst the hypothesis (STONE, GUEST and WILSON 1960) that borealis and flavomontana are derived from montana, but support THROCKMORTON'S (1978) conclusion of the early divergence of the former two species. This phylogenetic relationship supports our contention that the difference in the number of esterase genes with active alleles between borealis and montana is based on an increase in the number of genes coding for the alpha-esterases, rather than the retention in borealis of three genes with null alleles.