Delayed parasite clearance time observed in Southeast Asia provided the first evidence of Plasmodium falciparum resistance to artemisinins. The ex vivo ring-stage survival assay (RSA) mimics parasite exposure to pharmacologically relevant artemisinin concentrations. Mutations in the C-terminal propeller domain of the putative kelch protein Pf3D7_1343700 (K13) are associated with artemisinin resistance. Variations in the pfmdr1 gene are associated with reduced susceptibility to the artemisinin partner drugs mefloquine (MQ) and lumefantrine (LF). To clarify the unknown landscape of artemisinin resistance in Colombia, 71 patients with uncomplicated P. falciparum malaria were enrolled in a non-randomized observational study in three endemic localities in 2014–2015. Each patient's parasite isolate was assessed for ex vivo RSA, K13-propeller mutations, pfmdr1 copy number, and pfmdr1 mutations at codons 86, 184, 1034, 1042, and 1246, associated with reduced susceptibility, and 50% inhibitory concentration (IC50) for other antimalarial drugs. Ex vivo RSAs were successful in 56% (40/71) of samples, and nine isolates showed survival rates > 1%. All isolates had wild-type K13-propeller sequences. All isolates harbored either of two pfmdr1 haplotypes, NFSDD (79.3%) and NFSDY (20.7%), and 7.1% of isolates had > 1 pfmdr1 gene. In vitro IC50 assays showed that variable proportions of isolates had decreased susceptibility to chloroquine (52.4%, > 100 nM), amodiaquine (31.2%, > 30 nM), MQ (34.3%, > 30 nM), and LF (3.2%, > 10 nM). In this study, we report ex vivo RSA and K13 data on P. falciparum isolates from Colombia. The identification of isolates with increased ex vivo RSA rates in the absence of K13-propeller mutations and no positivity at day three requires further investigation.