The detection of cryptic species by biochemical methods indicates that within the phlebotomine fauna morphological data are not always adequate for species diagnosis. Cellulose acetate and polyacrylamide enzyme electrophoresis methods were compared for their effectiveness in identifying 13 species of Venezuelan phlebotomine sand flies and resolving alleles. Eight diagnostic loci unambiguously separated these 13 species of sand flies. Although acrylamide was as effective as cellulose acetate in species separation, differences were detected in the resolution of some alleles. Cellulose acetate identified more alleles at Ak and Fum, and resolved better at Pgm, whereas acrylamide identified more alleles at Gpi, Mdh, and Me. Therefore, erroneous species diagnoses may occur, if diagnostic loci detected by one technique are used by a second technique without adequate reference standards.