Background: Plasmodium vivax relapse resulting from hypnozoite activation, represents a significant mechanism for parasite persistence in the population. Primaquine (PQ), the drug of choice for eliminating hypnozoites, requires metabolic activation by Cytochrome P450-2D6 (CYP2D6). Genetic variations in CYP2D6 can alter PQ metabolism, potentially increasing the frequency of relapses. This study aimed to determine CYP2D6 polymorphisms in subjects with Plasmodium vivax under supervised chloroquine-primaquine treatment and explore their association with relapses as well as PQ plasma levels. Methods: CYP2D6 phenotypes and genotypes were successfully determined for 71 out of 78 patients included in the study. Nine polymorphisms (SNPs and indels) and gene copy number variation were analyzed. The association between the CYP2D6 phenotype, P. vivax relapse over six months follow-up, and PQ plasma levels were explored. Results: Most diplotypes (81.7%) were associated with normal (gNM-F) and ultrarapid (gUM) CYP2D6 metabolizers, while 18.3 % were associated with poor (gPM) and intermediate (gIM and gNM-S) metabolizers. On day 2, plasma levels of PQ were higher in individuals with poor/intermediate phenotypes compared to normal/increased phenotypes (660.4 ng/ml vs 313.5 ng/ml), although no statistical difference was found. The results were inconclusive about the association between relapse and poor/intermediate phenotype. Conclusion: The phenotypes of CYP2D6 associated with dysfunctional enzymatic activity (poor/ intermediate) are common in individuals with P. vivax in an endemic region of Colombia. Although no association between these phenotypes and relapse was found, further research is crucial due to their potential impact on radical cure treatment.