Two Calonectria species, C. henricotiae and C. pseudonaviculata, are recognized as the causal agents of boxwood blight, a recently emerged threat to the annual $1.3 million U.S. boxwood industry. The two species are difficult to distinguish based on morphology and pathogenicity, although some studies indicate that C. henricotiae, which is distributed only in Europe, exhibits greater fungicide tolerance. Fast, accurate, and inexpensive methods to detect and differentiate species allows accurate diagnosis of boxwood blight on plants as well as monitoring and detecting fungicide resistance. We designed primer pairs based on available sequences of three genes - calmodulin, histone H3, and ?-tubulin – and tested their ability to differentiate the two Calonectria species. Genomic DNA was extracted from 15 isolates of C. henricotiae and C. pseudonaviculata and was subjected to a real-time PCR amplification in the presence of fluorescent dye, followed by high-resolution melting analysis (HRMA). Primer pairs were identified that can differentiate the two species, and the 15 isolates were clustered into two distinct groups representing the two species, C. henricotiae (3) and C. pseudonaviculata (12). This HRMA is a fast high-throughput method to differentiate the boxwood blight pathogenic fungal species that doesn’t require gel or capillary analysis.