| Summary: | Despite their past importance as vectors of indigenous malaria, the species composition and spatial distribution of the members of the <i>Anopheles maculipennis</i> complex have been studied to a limited extent in the Netherlands. Therefore, this investigation focuses on the distribution of the members of this complex in the Netherlands, including <i>Anopheles daciae</i>, which has recently been found in countries bordering the Netherlands. In the framework of a national mosquito surveillance between 2010 and 2021, a total of 541 specimens of <i>An. maculipennis</i> s.l. were analyzed from 161 locations covering the entire territory. In addition, 89 specimens were analyzed from overwintering sites during the winter of 2020/2021. All individual mosquitoes were identified to species-level using Sanger sequencing of the ribosomal internal transcribed spacer 2. To characterize the habitat of <i>An. maculipennis</i> s.l. in the Netherlands, land cover use data was extracted in a 1 km buffer area around each finding location. For populations collected in summers between 2010 and 2021, the most frequent species was <i>An. messeae</i>, present in 88.19% of the locations, followed by <i>An. maculipennis</i> s.s. (11.80%), <i>An. atroparvus</i> (3.72%) and <i>An. daciae</i> (3.72%). <i>Anopheles daciae</i> was found in the southern inland areas of the country. Furthermore, <i>An. messeae</i> and <i>An. daciae</i> occurred in sympatry at overwintering sites. This study provides relevant information on the occurrence of species of the <i>Anopheles maculipennis</i> complex in the Netherlands, contributing to a better estimation of the risk of mosquito-borne disease in the country.
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