Leptospirosis outbreaks occur regularly in Argentina and other South American countries, but little is known about their epidemiological relationships. Application of new molecular tools, such as the Multiple-Locus Variable-number tandem repeat Analysis (MLVA) is limited by scant available data on regional strains. We have analyzed the genetic diversity of a collection of 31 strains of Leptospira interrogans isolated in Argentina during the past five decades from humans and animals, including a strain from an environmental source and another isolated from an opossum. Genotyping was performed by MLVA using the loci VNTR4, VNTR7, VNTR9, VNTR10, VNTR19, VNTR23 and VNTR31, as described by Majed et al. [Identification of variable-number tandem-repeat loci in Leptospira interrogans sensu stricto. J Clin Microbiol 2005;43:539-45]. Clustering analysis revealed eight distinct MLVA genotypes, with a dominant one, genotype A. Strains with this genotype were consistently isolated since 1960, representing 55% of the total strains and spanning an extensive geographical distribution. Other seven genotypes were less frequent, and only genotypes A and Hond Utrecht IV were isolated during the last decade. Different kinds of repeat blocks for each VNTR locus were identified by sequence analysis. VNTR copy number differences among genotypes always involved only one of these blocks. MLVA patterns obtained reveal the genetic diversity and relationships between strains, and constitute the framework for the genotyping of leptospires in the region.