Abstract Background Transition to dolutegravir-based regimens in resource-limited settings (RLS) requires prior understanding of HIV-1 integrase variants and conserved regions. Therefore, we evaluated integrase drug resistance mutations (DRMs) and conserved regions amongst integrase strand transfer inhibitor (INSTI)-naive patients harbouring diverse HIV-1 clades in Cameroon. Methods A cross-sectional study was conducted amongst 918 INSTI-naive patients from Cameroon (89 ART-naive and 829 ART-experienced patients). HIV-1 sequences were interpreted regarding INSTI-DRMs using the Stanford HIVdb v8.9-1 and the 2019 IAS–USA list. Amino acid positions with <1% variability were considered as highly conserved. Subtyping was performed by phylogeny. Results Overall prevalence (95% CI) of INSTI-DRMs was 0.8% (0.4–1.7), with 0.0% (0.0–4.0) amongst ART-naive versus 0.9% (0.5–1.9) amongst ART-experienced patients; P = 0.44. Accessory mutations (95% CI) were found in 33.8% (30.9–37.0), with 38.2% (28.1–49.1) amongst ART-naive versus 33.4% (30.4–36.7) amongst ART-experienced patients; P = 0.21. Of 288 HIV-1 integrase amino acid positions, 58.3% were highly conserved across subtypes in the following major regions: V75–G82, E85–P90, H114–G118, K127–W132, E138–G149, Q168–L172, T174–V180, W235–A239 and L241–D253. Wide genetic diversity was found (37 clades), including groups M (92.3%), N (1.4%), O (6.2%) and P (0.1%). Amongst group M, CRF02_AG was predominant (47.4%), with a significantly higher frequency (95% CI) of accessory mutations compared with non-AG [41.4% (36.8–46.0) versus 27.1% (23.3–31.2) respectively; P < 0.001]. Conclusions The low baseline of INSTI-DRMs (<1%) in Cameroon suggests effectiveness of dolutegravir-based regimens. In spite of high conservation across clades, the variability of accessory mutations between major circulating strains underscores the need for monitoring the selection of INSTI-DRMs while scaling up dolutegravir-based regimens in RLS.