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<i>Calonectria pseudonaviculata</i> is an emergent pathogen in the U.S. and one of the causal agents of the boxwood blight disease. The fungus was first reported in the U.S. in 2011 and rapidly spread to 10 additional states and three Canadian provinces. A second causal fungus, <i>C. henricotiae</i>, has not yet been reported in North America. Affected plants show necrotic spots on leaves, followed by defoliation, severe dieback and eventual plant death. The main challenges for the diagnosis of these pathogens are 1) the ubiquitous nature of the <i>Calonectria </i>species in soil and plants; 2) the possibility of latent infections; and 3) the unavailability of <i>Calonectria </i>genome sequences for identification of specific DNA markers. In this study, we sequenced and assembled draft genomes of the two boxwood blight fungi and three species of the <i>Nectriaceae </i>to identify novel and specific regions for the diagnosis of these pathogens. Based on different comparative genomic-based approaches, three LAMP assays were developed. The assays identified all <i>C. pseudonaviculata</i> and <i>C. henricotiae</i> isolates tested from representative genotypes from as little as 100 pg of DNA. No cross-reactions were observed with 11 additional <i>Calonectria </i>spp. and from other common fungal species and soil samples present in boxwood environments. The assays developed in this study will provide useful tools for monitoring and development of mitigation strategies towards the control of the boxwood blight fungi in the U.S.