Baseline genetic characterization of malaria vector populations provides critical data for evidence-based surveillance in persistent transmission foci. This pilot study generated preliminary genetic baseline data for Anopheles populations in the Menoreh Hills border region between Central Java and Yogyakarta provinces, Indonesia, addressing a key geographic gap in regional vector research. Adult female mosquitoes were collected from 3 houses with reported malaria cases in Ngadirejo Village using standardized entomological methods, including human landing, animal landing, and resting collections. Specimens were morphologically identified and molecularly characterized via ITS2 gene sequencing. Phylogenetic analyses were assessed using maximum likelihood methods, and genetic diversity indices were calculated to examine population structure. A total of 62 specimens representing 3 species were collected exclusively through animal landing collections: Anopheles vagus (48 specimens, 77.4%), Anopheles maculatus (9 specimens, 14.5%), and Anopheles kochi (5 specimens, 8.1%). An. kochi exhibited high haplotype diversity (Hd=0.709) with low nucleotide diversity (π=0.004), while An. maculatus showed lower haplotype diversity (Hd=0.480) and higher nucleotide diversity (π=0.026). Phylogenetic analysis revealed Purworejo specimens clustered with regional populations: An. kochi grouped within Clade I with Indonesian isolates; An. maculatus distributed across multiple clades; An. vagus formed a cohesive unit with other Indonesian populations. The exclusive success of animal landing collections in the Menoreh Hills highlands provides key methodological insights. This study offers essential baseline reference data, validates cost-effective genetic surveillance approaches, and supports future large-scale population connectivity studies across the Menoreh Hills malaria transmission complex.