We detected heartworm (Dirofilaria immitis) in 37.2% of 212 coyotes (Canis latrans) collected from 28 counties in Florida, US, between February 2010 and April 2014. Adult coyotes had a higher prevalence (45.6% of 103) than juveniles (29% of 80), and there was no significant difference in prevalence between adult male and female coyotes. Adults demonstrated a higher prevalence of heartworm in northern counties (56% of 91) than in southern counties (23.1% of 121) and a higher prevalence in urban areas (58.1% of 31) than in rural areas (33.3% of 63). There was no significant difference in mean body weight between infected and uninfected adult males or females. Intensity ranged from 1 to 144 (x̄ =21.29, SD=23.89, n=77). Mixed-sex heartworm infections were found in 88.9% of positive hosts, where the female-to-male ratio of worms was 1.14:1 (n=27). No pathologic changes attributable to heartworm infection were seen. A subsample of heartworms was confirmed to be D. immitis by targeted PCR amplification and DNA sequencing. Of 25 samples submitted for genotyping eight sequence haplotypes were found, all of which were distinct from all publicly available sequences derived from heartworms collected from domestic dogs (Canis lupus familiaris). These sequences revealed more polymorphisms than did previous samples of D. immitis, which provides a broader foundation for the possibility that the heartworms will develop resistance to anthelmintics.