We report Indian Plasmodium falciparum field isolates lacking both Pfhrp2 and Pfhrp3 genes. Lack of amplification of Pfhrp2, Pfhrp3 and their flanking genes in two P. falciparum field isolates of India. Row 1: Lanes 1 and 7=100bp marker, Lanes 6 and 12=negative control (distilled water), Lanes 2–5=Pfhrp2 gene, Lanes 8–11=Pfhrp3 gene. Row 2: Lanes 1 and 7=100bp marker, Lanes 6 and 12=negative control (distilled water), Lanes 2–5=upstream Pfhrp2 gene, Lanes 8–11=downstream Pfhrp2 gene. Display omitted ► Failure to express Pfhrp2 and Pfhrp3 due to gene deletion may contribute to variable performance of malaria RDTs. ► We report two Indian P. falciparum isolates lacking these genes. ► Genetic deletion of Pfhrp2 and Pfhrp3 may lead to false negative results in RDTs. Genetic polymorphisms in diagnostic antigens are important factors responsible for variable performance of rapid diagnostic tests. Additionally, the failure of antigen expression due to gene deletion may also contribute to variable performance. We report Indian Plasmodium falciparum field isolates lacking both Pfhrp2 and Pfhrp3 genes leading to false negative results of rapid diagnostic tests. The study highlights need to determine the prevalence of P. falciparum isolates lacking these genes in larger field populations in India.