There are 3 recognized chromosomal forms (B, E, K) in the taxon of Anopheles maculatus, 1 of the 8 members of the Anopheles maculatus group. Previous studies suggested that forms B and E are cytotypes of the species, but genetic characteristics of form K are unknown. The present study used crossing experiments, and polytene chromosomes of the ovarian nurse cell in F1 hybrids to show that form K is genetically distinct from forms B and E. In addition, postzygotic genetic incompatibility between form K and An. sawadwongporni, An. dravidicus, and An. pseudowillmori are demonstrated. In all crosses, hybrid males were sterile, with atrophied testes and accessory glands, or partially sterile with abnormal spermatozoa. The hybrid females showed varying degrees of atrophied ovaries. The ovarian nurse cell polytene chromosomes of the F1 hybrid females displayed ∼70 to almost complete asynapsis. The results provide clear evidence that form K should no longer be regarded as a cytotype of An. maculatus. The present study supports previous suggestions that form K represents another species of the Maculatus group. © 2008 by The American Mosquito Control Association, Inc.