These data consist of microsatellite genotype scores for all samples of Santa Ana sucker (Catostomus santaanae) used in the study. Scores represent the allele calls for each microsatellite locus (i.e. DNA fragment length containing the microsatellite repeats), with each locus containing two scores representing the two allele copies detected. Included are five tables: Full dataset (includes genotypes from all samples), Santa Clara River samples only (includes genotypes only from samples collected in the Santa Clara River), Convert File format key (explains the data file format), Population identifiers (translates the numerical population identifiers to actual collecting sites), CASA sampling points (one coordinate given for each general collection site). Whole specimens are accessioned at the Los Angeles County Museum of Natural History (catalog numbers 58475-58481). GenBank accession numbers for the DNA sequences are: MF918422 - MF918481. These data support the following publication: Richmond, J.Q., Backlin, A.R., Galst‐Cavalcante, C., O'Brien, J.W. and Fisher, R.N., Loss of dendritic connectivity in southern California's urban riverscape facilitates decline of an endemic freshwater fish. Molecular Ecology.