Lentic habitats (standing water, such as ponds and lakes) differ from lotic habitats (running water; streams and rivers) in their spatiotemporal persistence, with lentic habitats being more ephemeral in evolutionary time. This habitat instability is thought to select for dispersal, and several phylogenetic and macroecological studies have suggested that high rates of dispersal are more characteristic of lentic than lotic species. We tested this hypothesis using a comparative population genetic and phylogeographic approach based on mitochondrial DNA for 59 aquatic beetle species, sampled across Madagascar. Species were classified as lotic (n = 25), lentic (n = 25), or lotolentic (associated with both running and standing water; n = 9). Hierarchical population genetic structure (AMOVA), nucleotide diversity (π), and geographic structure were compared among habitat types. Lotic species had significantly greater population structure (Ф_ST = 0.55, hierarchical AMOVA) than lentic (Ф_ST = 0.13) and lotolentic (Ф_ST = 0.19) species using phylogenetic generalized least squares (PGLS) to correct for phylogeny. Body size was independent of habitat preference, and did not explain any of the intraspecific variation. A greater proportion of lotic species were endemic to Madagascar and lotic species had more pronounced geographic structure in their haplotype networks. The results indicate that dispersal is consistently lower among lotic species, independent of phylogenetic relatedness. This has macroevolutionary and biogeographical consequences for the freshwater fauna of this tropical biodiversity hotspot where remaining natural habitats are becoming increasingly isolated from one another.