Identifying patterns in genetic structure and the genetic basis of ecological adaptation is a core goal of evolutionary biology and can inform the management and conservation of species that are vulnerable to population declines exacerbated by climate change. We used reduced‐representation genomic sequencing methods to gain a better understanding of genetic structure among and within populations of Lake Tanganyika's two sardine species, Limnothrissa miodon and Stolothrissa tanganicae. Samples of these ecologically and economically important species were collected across the length of Lake Tanganyika, as well as from nearby Lake Kivu, where L. miodon was introduced in 1959. Our results reveal differentiation within both S. tanganicae and L. miodon that is not explained by geography. Instead, this genetic differentiation is due to the presence of large sex‐specific regions in the genomes of both species, but involving different polymorphic sites in each species. Our results therefore indicate rapidly evolving XY sex determination in the two species. Additionally, we found evidence of a large chromosomal rearrangement in L. miodon, creating two homokaryotypes and one heterokaryotype. We found all karyotypes throughout Lake Tanganyika, but the frequencies vary along a north–south gradient and differ substantially in the introduced Lake Kivu population. We do not find evidence for significant isolation by distance, even over the hundreds of kilometres covered by our sampling, but we do find shallow population structure.

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