Background The use of molecular techniques to detect malaria parasites has been advocated to improve the accuracy of parasite prevalence estimates, especially in moderate to low endemic settings. Molecular work is time-consuming and costly, thus the effective gains of this technique need to be carefully evaluated. Light microscopy (LM) and rapid diagnostic tests (RDT) are commonly used to detect malaria infection in resource constrained areas, but their limited sensitivity results in underestimation of the proportion of people infected with Plasmodium falciparum. This study aimed to evaluate the extent of missed infections via a community survey in Tanzania, using polymerase chain reaction (PCR) to detect P. falciparum parasites and gametocytes. Methods Three hundred and thirty individuals of all ages from the Kilombero and Ulanga districts (Tanzania) were enrolled in a cross-sectional survey. Finger prick blood samples were collected for parasite detection by RDT, LM and molecular diagnosis using quantitative 18S rRNA PCR and msp2 nPCR. Gametocytes were detected by LM and by amplifying transcripts of the gametocyte-specific marker pfs25. Results Results from all three diagnostic methods were available for a subset of 226 individuals. Prevalence of P. falciparum was 38% (86/226; 95% CI 31.9–44.4%) by qPCR, 15.9% (36/226; 95% CI 11.1–20.7%) by RDT and 5.8% (13/226; 95% CI 2.69- 8.81%) by LM. qPCR was positive for 72% (26/36) of the RDT-positive samples. Gametocyte prevalence was 10.6% (24/226) by pfs25-qRT-PCR and 1.2% by LM.