European honey bees in Australia are largely genetically isolated due to strict border controls. They contribute significantly to the economy through honey production and pollination, yet national efforts for their genetic improvement have historically been limited. This study estimated the genetic diversity of Australian bees using low-pass genome sequencing on 711 samples from 26 locations, provided by breeders participating in the national PlanBee project. Most genotypes were obtained from pooled drone samples. Results showed average observed heterozygosity (Ho: 0.20) was lower than the expected one. Bias tended towards low Ho, with some pooled drone samples conversely exhibiting unexpectedly high values. These high values were potentially attributed to multiple queen ancestries of the drone pool. Low FST values (0-0.07) between sampling locations indicated minimal population structure, likely due to gene flow through the exchange of genetic material within the PlanBee project. Findings suggest a need for broader sampling and better documentation of hive history and queen lineage.