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Background Clinical presentations of malaria in Ghana are primarily caused by infections containing microscopic densities of  Plasmodium falciparum , with a minor contribution from  Plasmodium malariae  and  Plasmodium ovale . However, infections containing submicroscopic parasite densities can result in clinical disease. In this study, we used PCR to determine the prevalence of three human malaria parasite species harboured by suspected malaria patients attending healthcare facilities across the country. Methods Archived dried blood spots on filter paper that had been prepared from whole blood collected from 5260 patients with suspected malaria attending healthcare facilities across the country in 2018 were used as experimental material.  Plasmodium  species-specific PCR was performed on DNA extracted from the dried blood spots. Demographic data and microscopy data for the subset of samples tested were available from the original study on these specimens. Results The overall frequency of  P. falciparum ,  P. malariae  and  P. ovale  detected by PCR was 74.9, 1.4 and 0.9%, respectively. Of the suspected symptomatic  P. falciparum  malaria cases, 33.5% contained submicroscopic densities of parasites. For all regions, molecular diagnosis of  P. falciparum ,  P. malariae  and  P. ovale  was significantly higher than diagnosis using microscopy: up to 98.7% (75/76) of  P. malariae  and 97.8% (45/46) of  P. ovale  infections detected by PCR were missed by microscopy. Conclusion Plasmodium malariae  and  P. ovale  contributed to clinical malaria infections, with children aged between 5 and 15 years harbouring a higher frequency of  P. falciparum  and  P. ovale , whilst  P. malariae  was more predominant in individuals aged between 10 and 20 years. More sensitive point-of-care tools are needed to detect the presence of low-density (submicroscopic)  Plasmodium  infections, which may be responsible for symptomatic infections. Graphical Abstract

Nationwide molecular surveillance of three Plasmodium species harboured by symptomatic malaria patients living in Ghana