In India, there are several malaria-endemic regions where non-falciparum species coexist with Plasmodium falciparum. Traditionally, microscopy and rapid diagnostic tests are used for the diagnosis of malaria. Nevertheless, microscopy often misses the secondary malaria parasite in mixed-infection cases due to various constraints. Misdiagnosis/misinterpretation of Plasmodium species leads to improper treatment, as the treatment for P. falciparum and Plasmodium vivax species is different, as per the national vector-borne disease control program in India. Blood samples were collected from malaria-endemic regions (Jharkhand, Madhya Pradesh, Chhattisgarh, Maharashtra, Odisha, Assam, Meghalaya, Mizoram and Telangana) of India covering almost the entire country. Molecular diagnosis of Plasmodium species was carried out among microscopically confirmed P. falciparum samples collected during a therapeutic efficacy study in different years. The polymerase chain reaction analysis revealed a high prevalence (18%) of mixed malaria parasite infections among microscopically confirmed P. falciparum samples from malaria patients that are either missed or left out by microscopy. Deployment of molecular tools in areas of mixed species infection may prove vital for accurate diagnosis and treatment of malaria. Further, it will help in achieving the goal of malaria elimination in India.