The Culex subgenus Phenacomyia is a small and poorly studied group of three mosquito species native to the American tropics. Here, we report the first detections of established populations of Culex (Phenacomyia) lactator Dyar & Knab in three counties of southern Florida. Culex lactator was first detected in May 2018 in southern Miami-Dade County, and, at this locality, was collected in subsequent years from 2018 to 2022 as both adults and immatures. Larvae and adults were subsequently collected in 2022, ∼175 km northwest of the initial locality at nine sites in Collier and Lee Counties. Identification of specimens collected in these counties as Cx. lactator is supported by molecular analysis and morphological characters of the adult female, male genitalia, and larva. The host associations and vector competence of Cx. lactator have not been extensively studied, and the public health implications, if any, of the addition of this species to Florida's mosquito fauna are unclear. These collections represent the first detections of Cx. lactator, or any Phenacomyia species, in the United States, adding to a trend in which detections of established populations of mosquito species from the American tropics in Florida appear to be increasing.