In Africa, the first Plasmodium falciparum artemisinin partial resistance mutation, was Kelch13 (K13) 561H - detected and validated at appreciable frequency in Rwanda in 2014. Surveillance to better define the extent of the emergence in Rwanda and neighboring countries is critical. We used novel liquid blood drop preservation combined with pooled sequencing to provide cost-effective rapid assessment of resistance mutation frequencies at multiple collection sites across Rwanda and neighboring regions in Uganda, Tanzania, and the Democratic Republic of the Congo (DRC). Malaria-positive samples (n=5,465) from 39 health facilities collected between May 2022 and March 2023 were sequenced in 199 pools. In Rwanda, K13 561H and 675V were detected in 90% and 65% of sites with an average frequency of 19.0% (0-54.5%) and 5.0% (0-35.5%), respectively. In Tanzania, 561H had high frequency in multiple sites. 561H appeared at 1.6% in Uganda. 561H was absent from the DRC, although 675V was seen at low frequency. Concerningly candidate mutations were observed: 441L, 449A, and 469F co-occurred with validated mutations suggesting they are arising under the same pressures. Other markers for decreased susceptibility to artemether-lumefantrine are common: P. falciparum multidrug resistance protein 1 N86 at 98.0% (63.3-100%) and 184F at 47.0% (0-94.3%) and P. falciparum chloroquine resistance transporter 76T at 14.7% (0-58.6%). Additionally, sulfadoxine-pyrimethamine-associated mutations show high frequencies. K13 mutations are rapidly expanding in the region further endangering control efforts with the potential of engendering partner drug resistance.