Blastomycosis due to Blastomyces dermatitidis and B. gilchristii is a notable cause of respiratory mycoses in North America with recurrent outbreaks. We developed a highly sensitive, specific, and reproducible Taqman duplex real-time PCR assay for the differentiation of B. dermatitidis and B. gilchristii. The new assay permitted retrospective analysis of Blastomyces cultures (2005 to 2019), and primary clinical specimens (2013-2019) from NY patients. Blastomyces dermatitidis was the causal agent for the majority of 38 cases while B. gilchristii was implicated in five cases; a rare finding reported from New York. The duplex real-time PCR assay will be useful for further understanding of ecology and epidemiology of blastomycosis caused by B. dermatitidis and B. gilchristii.