The sites of seven tRNA genes (Arg-2, Lys-2, Ser-2b, Ser-7, Thr-3, Thr-4, Val-3b) were studied by in situ hybridization. 125I-labeled tRNA probes from Drosophila melanogaster were hybridized to spreads of polytene chromosomes prepared from four Drosophila species representing different evolutionary lineages (D. melanogaster, Drosophila hydei, Drosophila pseudoobscura, and Drosophila virilis). Most tRNA loci occurred on homologous chromosomal elements of all four species. In some cases the number of hybridization sites within an element varied and sites on nonhomologous elements were found. It was observed that both tRNA(2Arg) and tRNA(2Lys) hybridized to the same site on homologous elements in several species. These data suggest a limited amount of exchange among different linkage groups during the evolution of Drosophila species.