HIV-1 genotyping and drug resistance tests are routinely performed in virology laboratories in some countries, aiding clinical management. In Istanbul, between January 2021 and March 2024, plasma samples from 1029 HIV-1-infected patients were analyzed using the NGS method, and mutation and drug resistance results were retrospectively evaluated alongside demographic data. Subtype B (54.4%) was most frequent in Turkish patients, while Subtype A1 (43.5%) was predominant among foreign nationals. The most common CRFs were CRF02_AG (3.8%) and CRF56_cpx (1.6%). According to the change in detection rates during the study period, Subtype B decreased, and Subtype A increased. The most frequent mutations detected were A62V (38.7%) and M184V (22.4%) for NRTIs; E138A (55.5%) and E138G (11.5%) for NNRTIs; M46I (33.3%) and M46L (25%) for PIs; and E92Q and G for INIs (total rate: 35.2%). Darunavir/ritonavir had the highest sensitivity rate, while resistance rates for NNRTIs and INIs increased over time. We anticipate that this study, in which we evaluate the routine use of an FDA-approved NGS kit alongside integrated bioinformatics data analysis and automated reporting software for the first time in Türkiye, will contribute to both national and international molecular epidemiological data and public health strategies by providing reliable results that align with international standarts.