Behavioural isolation from divergence in male advertisement calls and female preferences is hypothesized to cause genetic divergence and speciation in the Amazonian frogs Physalaemus petersi and P. freibergi, yet the importance of call variation and landscape features in genetic divergence is unresolved. We tested for correlations between genetic divergence at microsatellite loci and (1) call variables; and (2) landscape variables among 10 populations of these frogs. Genetic divergence was not correlated with geographical distance, rivers or elevation. There was a strong positive relationship, however, between genetic divergence and inter-population differences in one call variable, whine dominant frequency. Effective population sizes varied among sites (range = 15-846) and were often small, suggesting that genetic drift could influence call evolution. Evidence for fine-scale genetic structure within sites was also found. Our results support the hypothesis that behavioural isolation from divergence in male calls and female preferences causes genetic divergence and speciation.