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Various studies have shown the symbiotic association of Azospirillum brasilense with different grasses of nutritional importance. A high variability among the isolated strains has been observed. This is consistent with the genetic diversity that Azospirillum ha; shown in sugarcane isolates. However, if this is also the case in maize and sorghum isolates is unknown. In addition, it has also been reported that most of the isolates come from tropical regions where water is not limiting for maize and sorghum production. We analyzed the genetic variability of Azospirillum brasilense isolates obtained from arid zones of Northeast Mexico in maize and sorghum. Also, we characterized the phylogenetic relationship of five isolates selected from tier RFLP profiles with four Azospirillum species, as well as the phylogenetically related bacteria Rhizobium etli. The genetic variability was analyzed by PCR-RFLP of the gene 16S ADNr. The isolates were obtained from crops exposed to either rainfed or irrigated conditions. A high genetic variability was observed on the isolates, obtaining 14 different polymorphic bands in the study population with sizes ranging from 169 to 788 bp. RFLP analysis of the gene 16S ADNr showed the formation of two main clades of isolates: one group contained isolates coming from rainfed conditions, the other clade was composed by isolates from crops exposed to irrigation, and included the wild type strains. Results also showed that there were no differences between isolates either from maize or sorghum. The phylogenetic analysis based on the 16S ADNr sequences of five selected isolates, representative of the found diversity, showed the formation of two main clades, one of them containing only three isolates. The other clade with the two remaining isolates included the wild type strains. The separation of the isolates in both clades corresponded to the moisture regime they came from: either rainfed of irrigated conditions. This suggests that the differences observed in the groupings could be the result of diverse adaptation phenomena related to the water regime the crop grows in, and not to the grass species.