Achievement of treatment-free remission (TFR) after tyrosine kinase inhibitor (TKI) discontinuation in patients who show a durable deep molecular response (DMR) during TKI treatment of chronic myeloid leukemia in chronic phase (CML-CP) is a therapeutic goal; however, the prognostic factors that predict successful achievement of TFR are unclear. Previously, we reported that killer immunoglobulin-like receptor (KIR) and HLA polymorphisms are associated with achievement of a DMR. Here, we investigated the association between KIR and HLA polymorphisms and TFR. We conducted the POKSTIC (POlymorphisms of Killer immunoglobulin-like receptor, which affect Stop Tyrosine kinase Inhibitor in patients with Chronic myeloid leukemia) trial, a multicenter collaborative observational study that enrolled 76 patients with CML-CP. The median age was 63 years (interquartile range [IQR], 49-70). Of 76 patients, 42 (56.6%; 95% confidence interval [CI], 47.7-66.8 at 6 months) discontinued TKIs without molecular relapse; the median follow-up time for TFR was 24 months (IQR, 16-64). KIR genotyping and allele typing did not identify risk factors for molecular relapse; however, univariate and multivariate analysis identified the combination of KIR3DL1-HLA-Bw4 (an HLA-B allele) as an independent factor for a higher risk of molecular relapse (hazard ratio, 2.206; 95% CI, 1.112-4.376; P = .024). Notably, patients at higher risk of relapse had a significantly lower number of natural killer (NK) cells at TKI discontinuation than the other patients (CD16+/CD56+ NK cells: median 499.63 cells per μL vs 629.17 cells per μL, respectively; P = .049). Thus, KIR3DL1-HLA-Bw status reflects NK cell responses and is associated with TFR. The study is registered with the UMIN Clinical Trials Registry as #UMIN000041798.