Technical Abstract: The fungal pathogen Macrophomina phaseolina (Tassi) Goid. (=M. phaseoli [Maubl.] Ashby) causes charcoal rot disease in a vast number of plant species including soybean, cotton, sunflower and maize, resulting in significant economic losses. M. phaseolina exhibits apparent genetic adaptation to the plant hosts, however the molecular techniques used so far did not allow identification of possible genes involved in the host-pathogen interaction. Microsatellites or simple sequence repeats (SSR) are one of the most useful molecular tools with applications in population genetic studies. We developed 182 SSR markers for M. phaseolina; tested 24 isolates obtained from seven plant hosts, did cluster analysis of the fingerprinting data and analyzed the gene ontology of the sequences. Cluster analysis grouped most isolates by host of origin. SSR polymorphism in relation to gene ontology indicated that all essential biological processes of the pathogen had genetic variations depending on the host from which they were isolated. Based on those results, we performed additional tests on growth rate and copper resistance of the isolates identifying markers that could be related to these traits. The SSR markers developed here can be used to study population genetics of M. phaseolina, also for the fast identification of this species and to begin understanding the bases of genetic variations in this pathogen that may contribute to its host specificity. This knowledge could provide new tools for the control or management of this plant pathogen.