Macrophomina phaseolina (Tassi) Goid. is a soil-borne fungus with an extremely wide geographical distribution and large host range. Recently, increased incidence of the pathogen on diverse crop species has been reported worldwide, causing the significant economic losses in many field crops, including the sugar beet (Beta vulgaris L.). Charcoal root rot, caused by M. phaseolina is favored with higher temperatures (30-35°C) and low soil moisture, which often occurs during the summer months in Serbia and other South-Eastern European countries. In the study were used 58 M. phaseolina isolates, 56 from sugar beet, and one from maize and soybean. The objective of our study is to assess the genetic diversity of M. phaseolina isolates from sugar beet within local geographical area and estimate their genetic relationship, using SSR markers. The isolates were screened using 43 SSR primer pairs, out of which 13 were polymorphic. Genetic variation will be estimated by calculating the following parameters: number of polymorphic loci and their percentage, effective number of alleles per loci, expected heterozygosity and Shanon’s information index. For estimation of variance components among the isolates, analysis of molecular variance (AMOVA) will be performed. The genetic relatedness of M. phaseolina isolates will be established by cluster analyses performed on the matrix of genetic similarities.