The “Molobicus” hybrid breeding programme was initiated to improve tilapia growth performance in brackish water. The base population was created by backcrossing F1 Oreochromis niloticus GIFT strains × feral O. mossambicus to O. mossambicus and selective breeding conducted for performance in brackish water with two lines selected in extensive or intensive rearing conditions. A panel of ten diagnostic SNP markers was applied to estimate the species composition at different stages in the Molobicus programme including parental stocks, F1 and seventh generation fish from the selective lines. The O. aureus-specific markers tested revealed zero or negligible contribution from O. aureus to all the groups analysed. Feral O. mossambicus possessed an estimated 0.98 frequency of O. mossambicus-specific alleles, while GIFT samples had an estimated mean frequency of 0.88 O. niloticus-specific alleles. Hybrid F1 GIFT × feral O. mossambicus samples demonstrated close to 50:50 allele frequencies from O. niloticus and O. mossambicus for seven of the eight SNP loci tested. Analysis of the combined seventh generation Molobicus samples revealed a significant excess of O. niloticus alleles in six out of the eight SNPs tested, with this trend being more pronounced in the line selected in intensive culture conditions and showing increased body weight. PCR-based SNP assays such as these can be used to inform on the individual species contribution of fish stocks and provide tools for the genetic management of the tilapia species and future breeding programmes.