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.

Persistent malaria transmission remains a considerable challenge in Indonesia despite national elimination efforts, with the Menoreh Hills representing a residual transmission hotspot characterized by ecological and socio-behavioral complexity. This highland region, spanning the boundaries of the Kulon Progo, Purworejo, and Magelang districts presents a complex eco-epidemiological setting in which malaria transmission persists despite substantial reductions in incidence. Spatial analyses consistently reveal micro-foci and cross-district clusters, underscoring the structural and ecological interconnectedness of the Menoreh Hills, including Anopheles mosquito habitats. Anopheles mosquitoes exhibit ecological adaptability and behavioral plasticity that may contribute to residual transmission. This region features a heterogeneous landscape of secondary forests, agricultural lands, and rural settlements that foster diverse vector species and habitats, occupational exposure, and sustained human-vector contact associated with Plasmodium transmission. Hypnozoite-mediated relapse in P. vivax, prolonged gametocyte carriage in P. falciparum, and asymptomatic or submicroscopic infections may contribute to residual transmission. Surveillance is complicated by fragmented governance, variable funding, high human mobility across district borders, and logistical constraints imposed by rugged topography. This review emphasizes the necessity of strengthening integrated malaria surveillance systems that combine entomological, serological, genomic, and spatial data to enable early detection and targeted interventions. Cross-sectoral collaboration, community engagement, and adaptive, location-specific strategies addressing socio-ecological determinants alongside biological and environmental drivers are essential to interrupt persistent transmission in complex residual foci such as the Menoreh Hills.