Isozyme frequencies were compared in seven field collections of Anopheles minimus complex using starch gel electrophoresis. Mosquito collections were sampled from four districts in Kanchanaburi Province where malaria is endemic. From eight enzyme systems, nine loci and seven polymorphisms were detected, indicating limited genetic differentiation among the seven collections (F_ST = 0.061). The highest percent polymorphic loci were observed in Bong Ti Noi (BTN) Village (55.6%), whereas the least percent polymorphism was seen in Tha Kradan (TK) Village (22.2%). Comparing villages Pra Jedee (PJ) with Pu Teuy C (PTC) and Huai Khayeng (HK) with Pra Jedee (PJ), gene flow among collections varied from 3.72 to 62.25 reproductive migrants per generation. Among the seven collections, no correlation was seen between genetic and geographical distances (P > 0.05). Anopheles minimus (former species A) and Anopheles harrisoni (former species C) from Pu Teuy fit most closely in the same cluster, possibly indicating relatively recent divergence between taxa. The genetic and epidemiological ramifications of these findings are discussed.