This study assesses the genetic diversity of four different hatchery bred lines of Sydney rock oyster, Saccosstrea glomerata (SRO) after three, five and seven generations of mass selection using microsatellite DNA markers and mitochondrial cytochrome C oxidase subunit 1 sequences (COX1). This was achieved using seven newly developed microsatellite markers, along with three published loci. Considering the same number of samples in each line, the number of different alleles in each of the four hatchery lines ranged from 49 to 70 alleles from 10 loci, which was approximate 50% less than the 120 alleles found in the samples from the wild population. However, if we pool all our four hatchery lines together, then their total number of alleles was not significantly different from the wild population. Similar to the patterns found for the DNA microsatellite alleles, mtDNA haplotype numbers were considerably lower in each of the hatchery lines than in the samples from the wild population. Again, pooling the hatchery lines gave a total haplotype number not statistically significantly different from that in the samples from the wild. Considering together the DNA microsatellite alleles and the mtDNA haplotypes, we conclude that there was a substantial loss of genetic diversity within all lines separately over generations of mass selection, and that such mass selection was not sustainable in the long term. However, importantly, these data indicate that for oysters, if multiple independent mass selection lines are kept, then their combined genetic diversity may approach that of the ancestral line or wild population even after many generations of mass selection. These results for oysters, finding preservation of diversity among lines yet loss within them, are similar to recent findings for multiple banana shrimp lines; considering both species together it suggests that one relatively simple option suitable for some farms and industry to maintain genetic diversity during mass selection over many generations of selection is to subdivide their breeding nucleus and keep multiple different and independent lines over generations. It remains to be tested if this approach is general across many aquaculture species and circumstances.