Malaria vector control in pre-elimination settings faces challenges, including the detection and confirmation of infections in low-abundance Anopheles species. The circumsporozoite protein (CSP) enzyme-linked immunosorbent assay (ELISA) is widely used for vector control surveillance to incriminate vectors but can produce false positives in reported zoophilic-feeding mosquitoes. DNA extractions from CSP-ELISA homogenates are often used for molecular confirmation of Plasmodium falciparum infection, but failures may occur due to polymerase chain reaction (PCR) inhibitors or low DNA concentrations. This study evaluated a molecular hydrolysis probe real-time PCR assay using field-collected Anopheles mosquitoes from four African countries and compared infection detection results with those obtained using CSP-ELISA. The hydrolysis probe assay demonstrated comparable performance to CSP-ELISA despite detecting a low percentage of infections. These findings suggest that the hydrolysis probe assay provides a reliable alternative for confirming Plasmodium infections in field-collected mosquitoes. Overall, molecular approaches such as the hydrolysis probe assay may improve the accuracy of malaria vector surveillance and reduce reliance on DNA extraction from CSP-ELISA homogenates for confirmation.