Two populations of Hollandichthys multifasciatus from headwaters of adjacent river coastal basins in Paraná state, southern Brazil, were cytogenetically studied in order to evaluate intraspecific divergences. Both populations presented 2n = 50 chromosomes, divided into 14m 18sm 18st in the sample from Antonina and 14m 20sm 16st for the population from Guaraqueçaba. Analyses of active nucleolar organizer regions (Ag-NORs) and fluorescent in situ hybridization (FISH) with 18S rDNA probes revealed a single metacentric pair bearing marks at proximal positions on the short arms. The location of the 5S rDNA and GC-rich sites showed chromosomal divergence between both populations. Therefore, several population markers were detected, reflecting a population differentiation, possibly driven by the formation of Paranaguá Bay and Serra do Mar mountain range. Since the existence of a species complex in H. multifasciatus has been previously characterized by morphological, molecular, and karyotype population differentiation, this species needs to be taxonomically studied in detail.