For several years, there have been continuous reports of black flies in Mississippi with evidence of transmission of Leucocytozoon spp. This study was conducted to determine the presence and diversity of Leucocytozoon spp. in black flies captured throughout Mississippi. Thirty-three collections, consisting of 346 specimens, were made during the 2-yr period (2015–2016) at 10 locations around the state. In addition to these systematic biweekly collections, 46 specimens were taken in 10 collections at four additional sites from 2009 to 2014, as well as 388 specimens taken in 14 collections during a severe black fly outbreak during March and April of 2018. Out of 186 pooled samples, 21 samples were positive for haemosporidian DNA. Eighteen of those samples were identified as Leucocytozoon spp. A phylogenetic tree was constructed using the novel sequences along with existing sequences from the MalAvi database. Results showed several of the sequences constructed in this study had high divergence from the existing sequences from the database.