Unequivocal identification of phlebotomine sand flies is of crucial importance in epidemiological studies of leishmaniasis, because certain species may act as vectors, depending on behavior and physiology. For Lutzomyia whitmani, a major vector of American human cutaneous leishmaniasis in Brazil, an increasing number of studies have suggested the existence of a species complex. In the present work, we evaluated the genetic variability of L. whitmani populations from four Brazilian foci of that disease: Corte de Pedra, Ilhéus, Martinho Campos, and Serra de Baturité. Computational analysis of 85 characters, generated by RAPD-polymerase chain reaction, demonstrated high intrapopulational variability. Those characters led to sex discrimination in three of the populations, with the exception of Martinho Campos individuals, in which sex distinction was not complete. One and two interpopulational phenograms were obtained for females and males, respectively. A higher similarity was observed among the specimens from Ilhéus, Corte de Pedra, and Serra de Baturité, whereas the Martinho Campos population remained external to that cluster. These results, which are in partial accordance with a previous morphometric survey of L. whitmani from the same regions, provide additional evidence to support the existence of at least two spatial clusters of biogeographical populations of L. whitmani in Brazil.