The fungal pathogen, Calonectria pauciramosa, has caused serious diseases of many important plants worldwide. Understanding the genetic diversity and mating type distribution of this pathogen provides an essential step toward the development of disease control measures. In this study, we designed 15 polymorphic microsatellite markers by using genome sequences of two Ca. pauciramosa isolates having opposite mating type and from different countries. These markers were used to determine the genetic diversity of 145 isolates representing 13 different hosts (12 plant hosts residing in 12 genera, and soil) from 10 countries. In addition, mating type genes were amplified to investigate the reproduction mode of the pathogens in these populations by using mating type primers designed for Calonectria spp. Results revealed that a single dominant genotype, isolated from 11 plant genera residing in eight families, was present in seven countries across five continents. Only mating type MAT1-1 or MAT1-2 was amplified in each of the isolates, confirming that Ca. pauciramosa is heterothallic. Both mating types were detected in isolates from Eucalyptus in South Africa and Uruguay. The MAT1-2 phenotype was widely distributed in isolates from 12 different hosts (11 plant hosts and soil) collected in 10 countries. Overall, the results suggest that there has been substantial global movement of Ca. pauciramosa and that this has shaped its current population structure.