ABSTRACT. Maize landraces derived from tropical germplasm represent an important source of genetic variability, which is currently poorly understood and under-exploited by Brazilian crop breeding programs. The aims of our study were to a) estimate the genetic diversity across 48 varieties of maize landraces cultivated at different locations in the States of Rio Grande do Sul (RS) and Paraná (PR) by means of random amplified polymorphic DNA (RAPD), simple sequence repeat (SSR), and amplified fragment length polymorphism (AFLP) markers; b) cluster these varieties based on their genetic similarity estimates, and c) establish possible correlations between genetic similarity and germplasm collection sites. Maize landrace accessions were genotyped through the 30 RAPD, 47 SSR, and 25 combinations of AFLP primers. The results revealed high levels of variability across landraces within and between collection sites. AFLP analysis resulted in amplification of 762 polymorphic fragments and a polymorphic index of 40.3%, followed by RAPD with 335 fragments (81.9%) and SSR with 105 fragments (78.3%). The genetic similarity estimates of the investigated landraces ranged from 41 (SSR) to 74% (AFLP), and the amplitudes of these indices were notably similar between RAPD and SSR, as well as between AFLP and joint analysis. Regarding the RAPD and AFLP dendrograms, groups comprising accessions from RS prevailed, whereas SSR comprised varieties from both collection sites. Groups exclusive to RS or PR support the hypothesis that divergence between groups is possible owing to the fixation of regional adaptation alleles and to spatial barriers hindering genetic flow between locations.