Background: Malaria is one of the most serious parasitic diseases and the major health issue in tropical and subtropical countries. Objectives: This study aimed to investigate the type of infection of female Anopheles mosquitoes to sporozoites of Plasmodium species (Plasmodium vivax and Plasmodium falciparum), using nested polymerase chain reaction (PCR) in southeastern Iran. Methods: To determine the infections and Plasmodium species using the nested PCR in Sistan and Baluchestan Province (southeastern Iran), 360 female Anopheles mosquitoes were collected from the control villages of Chabahar and Konarak counties and examined by entomology check stations affiliated with Zahedan University of Medical Sciences in the second half of 2018. The mosquitos were caught manually from indoor places (humans and animals) as well as natural and artificial sites, i.e., pit-shelters. After DNA extraction, the molecular analysis was performed by nested PCR. Results: Of 200 species collected from Daregas, Owraki, and Bahu Kalat in Chabahar, four Anopheles stephensi (2%) were infected with P. vivax, and of 160 samples collected from the rural districts of Konarak, including Zarabad, Heyan, Islamabad, and Joholu, two An. stephensi (1.25%) were infected with P. falciparum. In this study, no mixed infection of Plasmodiumvivax and Plasmodium falciparum were observed. Conclusions: The present study showed that the only species infected with Plasmodium in the aforementioned regions was An. stephensi, which was the most frequent species of Anopheles among other species as well. The nested PCR could be the most suitable method for detecting Plasmodium parasites in malaria vectors, among other diagnostic methods.