ABSTRACT In recent years, crown and root rot of strawberry (Fragaria ×ananassa Duchesne ex Rozier) caused by Macrophomina phaseolina (Tassi) Goid. has affected strawberry production areas worldwide, and in developed countries its emergency has been attributed to the replacement of methyl bromide. The disease was reported in strawberry crop in Chile in 2013, in fields without fumigation. The use of resistant cultivars rises as an alternative to the management of this disease. The objective of this study was to perform a biological and molecular characterization of isolates obtained from two growing regions in Chile and Spain. A total of 35 isolates were characterized for mycelial growth at different temperatures and for chlorate sensitivity. Seven simple sequence repeat loci were used for genetic characterization. Differences were found between Chilean and Spanish isolates in both characterizations. The optimal temperature for mycelial growth was lower in Chilean than in Spanish isolates (30 and 35 °C, respectively). Meanwhile, Chilean isolates were more sensitive to chlorate. In terms of genetic characterization, Polymorphism Information Content (PIC) ranged from 0.38 to 0.85, two main groups were identified, the first group included Spanish isolates and the second group corresponded to Chilean isolates, results were supported by a population structure analysis. This study determined clear differences between two populations of Chilean and Spanish M. phaseolina isolates as causal agent of crown and root rot of strawberry.