A polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) assay developed for identification of five species of the Anopheles minimus Theobald group and a related mosquito species of the Myzomyia Series (Diptera: Culicidae) was applied to morphologically identified adult female specimens collected in Ratanakiri Province, north-eastern Cambodia. In addition to finding An. aconitus Dönitz, An. minimus species A and An. pampanai Büttiker & Beales, some specimens showed a new restriction banding pattern. Siblings of specimens that exhibited this new PCR-RFLP pattern were morphologically identified as An. culicifacies James sensu lato. Based on nucleotide sequences of the ribonuclear DNA internal transcribed spacer 2 region (ITS2) and the mitochondrial cytochrome oxidase I gene (COI), these specimens were recognized as An. culicifacies species B (sensu Green & Miles, 1980), the first confirmed record of the An. culicifacies complex from Cambodia. This study shows that the PCR-RFLP assay can detect species not included in the initial set-up and is capable of identifying at least seven species of the Myzomyia Series, allowing better definition of those malaria vector and non-vector anophelines in South-east Asia.