Background: Molecular HLA mismatch has shown greater biologic precision than conventional antigen mismatch for estimating alloimmune risk after kidney transplantation. Prior work in an ethnically diverse steroid-sparing cohort demonstrated that increasing molecular mismatch was associated with early de novo donor-specific antibody (dnDSA) development, but follow-up was limited to the early post-transplant period. We performed a longitudinal extension study to evaluate whether the same mismatch framework remains associated with cumulative dnDSA development and rejection through 24 months. Methods: We analyzed a patient-level cohort of 630 kidney transplant recipients generated as a scaled longitudinal extension of a published 3-month study. Baseline variables included sex, recipient ethnicity, transplant type, graft number, induction/maintenance regimen, total ABDR antigen mismatch, and three molecular mismatch metrics: HLA-Matchmaker antibody-verified (AbVer) eplet load, HLA-EMMA solvent-accessible (SolAcc) amino-acid mismatch load, and PIRCHE-II peptide mismatch load. Outcomes were cumulative dnDSA at 3, 6, 12, 18, and 24 months, biopsy occurrence, and cumulative rejection through 24 months. Logistic regression and receiver operating characteristic analysis were used to compare mismatch measures. Results: In the 630-patient cohort, cumulative dnDSA incidence rose from 77/630 (12.2%) at 3 months to 91/630 (14.4%) at 6 months, 119/630 (18.9%) at 12 months, 134/630 (21.3%) at 18 months, and 149/630 (23.7%) at 24 months. Cumulative rejection increased from 44/630 (7.0%) to 93/630 (14.8%) over the same intervals. At 3 months, 54 of 77 dnDSA events were persistent and 23 were transient. By 24 months, 72 additional late dnDSA events were modeled, most frequently between 6 and 12 months. Higher 24-month dnDSA burden was associated with higher median AbVer, SolAcc, and PIRCHE-II loads. In multivariable analysis, each mismatch metric remained associated with cumulative 24-month dnDSA: ABDR mismatch odds ratio (OR) 1.385, HLA-Matchmaker OR 1.152, HLA-EMMA OR 1.037, and PIRCHE-II OR 1.004 (all P < 0.001). HLA-EMMA showed the strongest single-metric discrimination for 24-month dnDSA (area under the curve [AUC] 0.761), compared with 0.733 for HLA-Matchmaker, 0.639 for PIRCHE-II, and 0.612 for ABDR mismatch. Rejection by 24 months occurred in 14.8% overall but was concentrated among recipients with persistent or late dnDSA. Conclusions: In this longitudinal extension, higher molecular mismatch burden remained strongly associated with cumulative dnDSA development and rejection through 24 months. HLA-EMMA and HLA-Matchmaker outperformed conventional antigen mismatch for dnDSA discrimination, supporting further prospective evaluation of molecular mismatch-informed surveillance and immunosuppression stratification in ethnically diverse steroid-sparing kidney transplant programs.