Abstract. Barriers to gene flow between Pacific and Atlantic coast populations of Anopheles albimanus were reported in an earlier study of variation in the intergenic spacer of the nuclear ribosomal DNA. We examined the distribution of mitochondrial DNA haplotypes among A. albimanus populations to test for gene flow barriers with an independent genetic marker. A region of the NADH dehydrogenase subunit 5 gene was amplified by the polymerase chain reaction (PCR) in 1,105 mosquitoes collected from 16 locations in Guatemala and in single collections from Mexico, Honduras, Nicaragua, Costa Rica, Panama, Colombia, and Venezuela. The PCR products were tested for variation using single strand conformation polymorphism analysis and 45 haplotypes were detected. Haplotype frequencies did not vary between coasts in Guatemala. Populations within ϳ200 km of one another were panmictic. However, at distances Ͼ 200 km, F ST and geographic distances were correlated suggesting that populations are isolated by distance.Anopheles albimanus is one of the major vectors of malaria in Central America, the Caribbean, and coastal regions of northern South America. It feeds more readily on animals than humans, 1-3 and is generally exophilic in host-seeking and feeding behavior. [4][5][6] Sporozoite rates are generally low, with the greatest reported rate at only 1.6% (1 of 63). 7 However, these behavioral characteristics and vector competence estimates vary greatly among A. albimanus populations. 8 We have been testing for the presence of cryptic species within A. albimanus because of the strong historical precedence for the existence of cryptic, isomorphic Anopheles species.In an earlier study of Guatemalan A. albimanus populations, we observed a high degree of variation both in the frequency and copy number of intergenic spacer (IGS) length variants among individual A. albimanus. 9 Regardless of the collection site, most of the variation either in frequency or intensity of IGS variants was detected among mosquitoes within a population. The next largest component of genetic differentiation was detected between populations on the Pacific and Atlantic coasts of Central America. In contrast, populations from the Atlantic coast of Central America and from South America had similar IGS patterns. We suggested that the many mountain ranges that run through northern Central America could form barriers to gene flow among Atlantic and Pacific coast populations. However, extreme local variation in rDNA IGS has been found in many population studies of other insect species and is consistent with a model whereby molecular drive rapidly gives rise to unique spacer patterns in populations and then drives them to high frequencies. [10][11][12][13] In the case of A. albimanus, molecular drive probably occurs through unequal crossing-over and gene conversion within and among the 100-basepair (bp) repeat units constituting the IGS. 14 In the present paper, we test the inferences arising from our earlier rDNA study using an independent marker: variation in t...