Zebrafish (Danio rerio), a small freshwater fish that originates from India, Bangladesh, Nepal, Bhutan and northern Myanmar, have been widely used as a model organism for studies of developmental biology and genetics. The current study aimed to determine the origin of South African pet shop stock that are currently being used to establish a laboratory population founded from diverse sources available locally. Zebrafish DNA was extracted from 65 specimens housed at the University of the Free State (UFS) Department of Genetics. For phylogenetic analysis, cytb sequences were generated from all samples. A further 178 sequences were downloaded from the GenBank database, including sequences of an outgroup species (Danio kyathit). Five microsatellite markers were used to further assess the genetic diversity of the UFS zebrafish specimens. A maximum likelihood analysis was performed for the cytb data. Results of the phylogenetic analyses divided the sequences into three major genetic groups, which was congruent with a previous study on laboratory zebrafish provenance. The SA pet shop fish grouped with the strains from the northern and north-eastern regions of India. High levels of microsatellite genetic diversity were observed for the pet shop sourced population, correlating to what has previously been observed in zebrafish. These results can be used to guide the future development of laboratory strains suited to the needs at the UFS.