Emergence of Microvariants of African Swine Fever Virus Genotype II in the Asia–Pacific

African swine fever virus (ASFV) is a highly stable DNA virus showing little genetic variation among genomes. This genetic stability has often posed challenges in tracking variants and identifying potential transmission chains as ASFV spreads into new regions. While mutations within individual sequenced genes are infrequent, the application of whole-genome sequencing enables the identification of neutral and functional mutations across the entire genome, contributing to a deeper understanding of ASFV evolution and epidemiology. In this study, we sequenced whole genomes from 25 ASFV positive samples collected from Vietnam, Timor-Leste and Papua New Guinea (PNG). We classified mutations across ASFV genes and non-coding regions, while identifying mutations that may act as candidates for geographic-based population genetic structuring. Overall, ASFV samples showed >99.8% genetic similarity to the Georgia 2007 isolate reference genome. Nonetheless, emergent genetic clusters rooted in geographic location were apparent. Multiple nonsynonymous mutations were found in sequenced genomes, often unique to ASFV collected within only one studied country. Of the observed mutations, four were found within genes of known function (E199L, CP80R, B962L and B602L), with the latter (B602L) being a novel mutation within a known targeted marker gene for conventional genotyping of the virus. This work highlights the benefits of incorporating whole-genome sequencing into ongoing ASFV surveillance to better reflect the natural population genetic structuring within the species as it has spread across the Asia–Pacific region. Additionally, this work highlights the value of whole-genome sequencing as a tool for actively monitoring emergent variants displaying functional divergence in key genes associated with ASFV proliferation and host selection.