Background Young key populations (YKP) contribute to the burden of HIV in Nigeria and are a priority population for oral pre-exposure prophylaxis (PrEP). However, their uptake of PrEP remains low. We assessed the main barriers to PrEP uptake and the variation among YKP (15–24 years) in Nigeria. Methods This study was a secondary data analysis of the 2020 Integrated Biological & Behavioural Surveillance Survey conducted among key populations (KP), including female sex workers (FSW), men who have sex with men (MSM), people who inject drugs (PWID), and transgender people (TG), in 12 states in Nigeria. A closed-ended question asking the main reason for not taking PrEP among KP who had never taken PrEP was included in the surveillance questionnaire. We collapsed the responses into six barrier themes. Using multinomial logistic regression analysis, we examined the association between the barriers (dependent variable) and KP group (independent variable), controlling for age, educational attainment, religion, marital status, employment status, and geopolitical zone. Results A total of 1776 YKP were included in this study. The most cited barriers by KP group were: lack of access (28.3%) and fear of side effects (28.3%) by FSW; lack of interest (37.1%) by MSM; low risk perception (65.5%) by PWID; and lack of access (34.4%) by TG. The odds of reporting fear of side effects, lack of access, lack of interest, and nonspecific/others reasons were significantly different by KP group. Conclusions The barriers limiting the uptake of PrEP among YKP vary by KP group. Our results highlight the need for KP-specific interventions to improve the uptake of PrEP among YKP in Nigeria.