OBJECTIVE: To describe the genetic diversity of Plasmodium vivax isolates from different areas in the Brazilian Amazon using 11 polymorphic microsatellites and to evaluate the correlation between microsatellite variation and repeat array length. METHODS: Microsatellites with variable repeat units and array lengths were selected using in silico search of the P. vivax genome. We designed primers and amplified the selected loci in DNA obtained from patients with P. vivax acute infections. RESULTS: Positive correlation between repeat array length and microsatellite variation was detected independently of the size of repeat unit (di, tri, or tetranucleotide). We used these markers to describe the genetic variability of P. vivax isolates from four geographic regions of the Brazilian Amazon. Substantial variability was observed among P. vivax isolates within populations, concurrent with high levels of multiple-clone infections and high linkage disequilibrium. Overall, structured populations were observed with moderate to high genetic differentiation. CONCLUSION: The markers studied are useful tools for assessing population structure of P. vivax, as demonstrated for Brazilian populations and for searching for evidence of recent selection events associated with different phenotypes, such as drug resistance.