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Emergence of HIV drug resistance poses a serious risk of inactivity to all currently approved antiretroviral drugs. Profiles of HIV drug resistance mutations (HIVDRM) and virological failure (VF) are not extensively studied in Tanzania. This study aimed to determine HIVDRM and predictors of VF in HIV-infected individuals failing first-line HIV drugs in Moshi, Northern Tanzania. A case-control study was conducted at Kilimanjaro Christian Medical Centre, Mawenzi, Pasua and Majengo health facilities with HIV-care and treatment clinics from October, 2017 to August, 2018. Cases and controls were HIV-infected individuals with VF and viral suppression (VS) respectively. HIV-1 reverse transcriptase and protease genes were amplified and sequenced. Stanford University’s HIV drug resistance database and REGA subtyping tool 3.0 determined HIVDRM and HIV-1 subtypes respectively. Odds ratios (OR) with 95% confidence interval (95% CI) investigated predictors of VF. P-value < 5% was considered statistically significant. A total of 124 participants were recruited, of whom 63 (50.8%) had VF, 61 (49.2%) had VS and 82 (66.1%) were females. Median [IQR] age and duration on ART were 45 [35–52] years and 72 [48–104] months respectively. Twenty-five out of 26 selected samples from cases were successfully sequenced. Twenty-four samples (96%) had at least one major mutation conferring resistance to HIV drugs, with non-nucleoside analogue reverse transcriptase inhibitor (NNRTI)-resistance associated mutations as the majority (92%). Frequent NNRTI-resistance associated mutations were K103N (n = 11), V106M (n = 5) and G190A (n = 5 …