ABSTRACT. Most studies of diversity and genetic structure in neotropical fish have focused on commercial species from large rivers or their reservoirs. However, smaller tributaries have been identified as an important alternative migratory route, with independent pools of genetic diversity. In this context, the present study aimed to evaluate genetic diversity and structure in five neotropical fish species from a region of Laranjinha River in the upper Paraná River basin. PCR-RAPD (random amplified polymorphic DNA) markers were used to characterize around 40 individuals of each species distributed upstream and downstream of Corredeira Dam that interrupts the river. The descriptive index of genetic diversity (P = 30.5-82%; H E 0.122-0.312) showed that the populations have acceptable levels of genetic diversity. The values for Nei's genetic distance (D N min 0.0110 and max 0.0306) as well as the genetic structure index and the analysis of molecular variance (AMOVA, ϕ ST min 0.0132 and max 0.0385) demonstrated low, but significant levels of genetic structure. Bayesian analysis of assignment found two k clusters, including several individuals with mixed ancestry for all populations from the five species analyzed. These findings along with historical data on rainfall and the low dimensions of the dam studied here support the hypothesis that periodic floods enable the transit of individuals between different localities mitigating the differentiation process between populations.