One modification of the primitive karyotype of the Drosophilidae is the absence of dot chromosomes. The origin of this modification is diverse. In some cases, the fate of the dot chromosomes can be directly inferred from cytogenetic analysis but in other cases a genetic or a combined molecular and cytogenetic analysis is needed, as occurs in Drosophila willistoni and Scaptodrosophila lebanonensis. We determined the location of four dot chromosome sequences in D. willistoni and S. lebanonensis using in situ hybridization. Drosophila melanogaster and D. virilis, which possess dot chromosomes, were used as a control. The in situ hybridization results show that dot chromosome genes of D. melanogaster and D. virilis are closely linked in chromosome 3 of D. willistoni and in chromosome X of S. lebanonensis. These results suggest an autosome-dot fusion in D. willistoni and an X-dot fusion in S. lebanonensis, two different paths in the evolution of dot chromosomes.