Malaria is one of the most severe public health problems in Ghana. In developing countries such as Ghana, with high of prevalence of malaria, the procedures for diagnoses and detection is limited in technological depth and for that reason most of these parasites; Plasmodium ovale, Plasmodium malariae, Plasmodium vivax and Plasmodium knowlesi are not routinely screened for; as such this study seeks to determine the prevalence of Plasmodium species among humans and monkeys at Mole National Park in Northern Ghana. A total sample size of 217 comprising 214 human subjects and 3 baboons were recruited in this study conducted at Mole National Park. Data including; age, marital status, gender, occupation, knowledge on malaria and educational background information were collected. Blood samples were taken from both humans and baboons for the malaria testing using RDT, Microscopy and PCR to detect Plasmodium falciparum, Plasmodium ovale, Plasmodium malariae, Plasmodium vivax and Plasmodium knowlesi. The mean age of the study participants was 24.2±15.2. All the 3 baboon subjects were negative on RDT but one (1) out of the three was positive on microscopy. That notwithstanding, the same microscopy positive baboon subject was Pan-Plasmodium positive on PCR but was negative for the tested Plasmodium falciparum, Plasmodium ovale and Plasmodium malariae. The study recorded prevalence of plasmodium species using RDT and microscopy respectively to be 22.9% and 18.2%. The prevalence of Plasmodium falciparum, Plasmodium ovale and Plasmodium malariae among the human participants from this study were 39.9%, 12.6% and 18.6% respectively. Mixed infections were identified in 7.5% of the human participants where 4.2% was Plasmodium falciparum/Plasmodium ovale, 2.8% was falciparum/malariae mixed infection and 0.5% malariae/ovale mixed infection. Sociodemographics, knowledge on malaria and interaction with monkeys of study participants did not show any significant association with malaria infection. Prevalence of malaria parasites was high among the participant, which poses threat to reduction of malaria in the country.