Malaria continues to affect over 200 million individuals every year, especially children in Africa. Rapid and sensitive detection and identification of Plasmodium parasites is crucial for treating patients and monitoring of control efforts. Compared to traditional diagnostic methods such as microscopy and rapid diagnostic tests (RDTs), DNA based methods, such as polymerase chain reaction (PCR) offer significantly higher sensitivity, definitive discrimination of Plasmodium species, and detection of mixed infections. While PCR is not currently optimized for routine diagnostics, its role in epidemiological studies is increasing as the world moves closer toward regional and eventually global malaria elimination. This study demonstrates the field use of a novel, ambient temperature-stabilized, multiplexed PCR assay in a small hospital setting in Sierra Leone.