Entomological surveys were conducted in the malaria-endemic southwestern region of Tajikistan to establish which species of Anopheles may be responsible for malaria transmission. The head—thorax portions of 2,213 wild-caught Anopheles females—Anopheles superpictus Grassi (n = 1,292), Anopheles pulcherrimus Theobald (n = 376), Anopheles hyrcanus (Pallas) (n = 544), and Anopheles claviger (Meigen) (Diptera: Culicidae) (n = 1)—were tested for the presence of Plasmodium sporozoites using enzyme-linked immunosorbent assays. Four females of An. superpictus (three captured when landing on humans and one resting indoors) and one female of An. hyrcanus (captured when landing on a human indoors) were positive for Plasmodium vivax (Haemosporida: Plasmodiidae) VK-210. The infected females of An. superpictus were captured in the Hamadoni and Yovon districts of the Khatlon province, and the single infected female of An. hyrcanus was captured in the Jilikul district of the Khatlon province.