Understanding genetic diversity and population structure is essential for the effective conservation and management of threatened freshwater fishes. Murray cod (Maccullochella peelii), one of Australia's largest freshwater fishes, has undergone significant population declines due to river regulation, habitat degradation, and historical overexploitation. In this study, we assessed the population genetic structure and genetic diversity of Murray cod across the regulated lowland Lachlan River. Using genome-wide single nucleotide polymorphism (SNP) markers generated by Diversity Arrays Technology Sequencing (DArTseq), our analyses focused on both spatial and temporal patterns of genetic variation. Population genomic analyses revealed genetic differentiation between fish collected from the mid Lachlan (upstream of Lake Brewster) and lower Lachlan (downstream of Lake Brewster) in 2025, indicating spatial population structuring within the river. In addition, samples collected from the lower Lachlan in 2015 formed a distinct genetic cluster from those collected at the same locations in 2025, suggesting temporal changes in population composition. Lower Lachlan samples also exhibited slightly lower genetic diversity (observed heterozygosity, Ho = 0.329) than samples from the mid Lachlan (Ho = 0.339). Although identifying the underlying drivers of this temporal genetic differentiation is beyond the scope of this study, the observed pattern may reflect the combined effects of stocking, limited natural recruitment, and the major hypoxic blackwater event in 2016, which caused substantial mortality of large-bodied native fish. Collectively, this study provides new insights into the spatial and temporal genetic dynamics of Murray cod in the Lachlan River and highlights the importance of long-term genomic monitoring for informing adaptive conservation and stocking strategies in regulated river systems.