Among novel compounds under investigation as potential new antimalarial drugs are three independently developed inhibitors of PfATP4: SJ733, PA92, and KAE609 (cipargamin). We assessed ex vivo sensitivities to these compounds of fresh P. falciparum isolates from Tororo and Busia districts in Uganda from 2016-2019. We then characterized genotypes of parasites with varied sensitivity to the PfATP4 inhibitors. Ex vivo drug sensitivity was determined with 72 h growth assays utilizing SYBR green. We observed a range of sensitivities; geometric mean IC50s were 58.5 nM for SJ733, 8.5 nM for PA92, and 0.46 nM for KAE609. Sensitivities for the three PfATP4 inhibitors correlated: rs for correlation 0.64 for PA92 and SJ733, 0.51 for PA92 and KAE609, and 0.36 for SJ733 and KAE609. Sequencing of PfATP4 for 218 isolates demonstrated many non-synonymous SNPs in single and mixed (52%) infections. The most frequent mutations were G1128R (66% of isolates), Q1081K/R (65%), G223S (23%), N1045K (14%), D1116D/Y/N (13%), T507S (7%), and G1031S (6%). We found associations between G223S and decreased sensitivity to SJ733 (mean IC50 70.6 nM for WT, 72.1 nM for mixed, 92.6 nM for mutant), PA92 (IC50 9.7 nM for WT, 11 nM for mixed, 14.9 nM for mutant) and KAE609 (IC50 0.5 nM for WT, 0.5 nM for mixed, 0.7 nM for mutant); for all comparisons of WT versus mutant p<0.05. Interestingly, previous studies showed that incubation of a laboratory strain with an analog of KAE609 selected for resistant parasites with a mutation (G223R) at the same allele. Our results indicate that malaria parasites circulating in Uganda have a number of polymorphisms in PfATP4, and that at least one common mutation is associated with decreased sensitivity to some PfATP4 inhibitors now under development.