A previously established species-diagnostic PCR assay was used to determine the species composition and distribution of sibling species of the Anopheles maculipennis complex in north-eastern Poland, which was historically affected by malaria. Of all the 1120 mosquitoes of An. maculipennis complex (1002 adults and 118 larvae) identified by molecular means, all were either An. messeae/daciae or An. ... [Show full abstract] maculipennis s.s. with the former dominant in all collection localities. Their halophilic sister taxon An. atroparvus, previously collected along the Baltic Sea coast was not found. New records of An. maculipennis s.s. and An. messeae were located in 9 UTM squares respectively and these species were new for Tuchola Forests (Bory Tucholskie) and for the district of Iława Lakeland (Pojezierze Iławskie). An. messeae is a new species also for Masurian Lakeland (Pojezierze Mazurskie).