Background: The assessment of geographical heterogeneity of HIV among men who have sex with men (MSM) and people who inject drugs (PWID) can usefully inform targeted HIV prevention and care strategies. Objective: We aimed to measure HIV seroprevalence and identify hotspots of HIV infection among MSM and PWID in Nigeria. Methods: We included all MSM and PWID accessing HIV testing services across 7 prioritized states (Lagos, Nasarawa, Akwa Ibom, Cross Rivers, Rivers, Benue, and the Federal Capital Territory) in 3 geographic regions (North Central, South South, and South West) between October 1, 2016, and September 30, 2017. We extracted data from national testing registers, georeferenced all HIV test results aggregated at the local government area level, and calculated HIV seroprevalence. We calculated and compared HIV seroprevalence from our study to the 2014 integrated biological and behavioural surveillance survey and used global spatial autocorrelation and hotspot analysis to highlight patterns of HIV infection and identify areas of significant clustering of HIV cases. Results: MSM and PWID had HIV seroprevalence rates of 12.14% (3209/26,423) and 11.88% (1126/9474), respectively. Global spatial autocorrelation Moran I statistics revealed a clustered distribution of HIV infection among MSM and PWID with a <5% and <1% likelihood that this clustered pattern could be due to chance, respectively. Significant clusters of HIV infection (Getis-Ord-Gi* statistics) confined to the North Central and South South regions were identified among MSM and PWID. Compared to the 2014 integrated biological and behavioural surveillance survey, our results suggest an increased HIV seroprevalence among PWID and a substantial decrease among MSM. Conclusions: This study identified geographical areas to prioritize for control of HIV infection among MSM and PWID, thus demonstrating that geographical information system technology is a useful tool to inform public health planning for interventions targeting epidemic control of HIV infection.