A total of 4 471 samples, collected between January 2022 and August 2024 from healthcare facilities, and during active surveillance in malaria-eliminating districts in KwaZulu-Natal and Mpumalanga provinces, were assessed for drug (mutations in the kelch13, crt, mdr1, dhfr, and dhps genes) and diagnostic (hrp2/3 gene deletions) resistance markers using PCR and sequencing (Sanger and/or targeted amplicon) protocols. The gene discussed is DHFR; the disease is malaria.