Malaria remains a major public health concern worldwide, with Anopheles mosquitoes serving as its primary vectors. The widespread use of synthetic insecticides in malaria control programs has resulted in the development of insecticide resistance in these mosquitoes, endangering control efforts. This study investigates metabolic resistance mechanisms in wild-caught Anopheles spp. from three regencies in Indonesia (Pesawaran, Pangandaran, and Purworejo), with an emphasis on enzymatic activity and ace-1 gene mutations. Anopheles mosquitoes were collected using human landing capture (HLC) and cow-baited collection (CLC). Biochemical assays were performed to evaluate the activity of acetylcholinesterase (AChE), glutathione S-transferase (GST), and oxidase. Additionally, ace-1 gene mutations were analyzed using PCR-RFLP. Seven Anopheles species were identified. Statistical analysis revealed significant differences in mosquito distribution across the regencies based on HLC (P < 0.05). All Anopheles populations exhibited significantly elevated AChE and GST activity (P < 0.05), suggesting strong metabolic resistance. Anopheles barbirostris from Pesawaran regency exhibited heterozygous ace-1 gene mutations (194, 120, and 74 bp bands), classifying it within AChE resistance group A. The findings confirm widespread metabolic resistance in Anopheles mosquitoes across Indonesia, which is most likely caused by chronic insecticide exposure. Continuous monitoring of insecticide resistance and its underlying mechanisms is critical to sustain malaria control and eradication efforts in Indonesia. © 2025 Elsevier B.V.