Pathogenic and genetic characterizations of 96 isolates of Macrophomina phaseolina from Mexico and other countries were carried out in order to define the pathogenic and genetic patterns of diversity and to gain insights into the pathogenic and genetic specialization in this fungus. Isolates were collected from different hosts in Mexico (23 isolates), Italy, Australia, Japan, Argentina, USA, Colombia and Brazil (73 isolates). Pathogenicity was evaluated in seeds of two Phaseolus vulgaris L. cultivars under in vitro conditions, while genotype was determined on the basis of amplified fragment-length polymorphisms (AFLPs). The most frequent pathotypes were 4095 (15 isolates) and 0 (nine isolates), while 59 isolates had a unique pathotype (61%). Cluster analysis showed two contrasting groups of isolates on the basis of pathogenicity, where one group mainly included the most aggressive isolates from Mexico and Colombia. The AFLP analysis produced 418 amplified products and 92.8% were polymorphic. Cluster analysis showed no clear association between AFLP genotype and geographical origin. When subgroups of isolates were re-analysed, we found a clear differentiation between Mexican and non-Mexican isolates. Results confirmed the significant pathogenic and genetic diversity of Macrophomina and showed a clear differentiation between Mexican isolates from all others.