Abstract Background Evidence on the distribution of pre-treatment HIV-1 drug resistance (HIVDR) among risk groups is limited in Africa. We assessed the prevalence, trends and transmission dynamics of pre-treatment HIVDR within and between MSM, people who inject drugs (PWID), female sex workers (FSWs), heterosexuals (HETs) and perinatally infected children in Kenya. Methods HIV-1 partial pol sequences from antiretroviral-naive individuals collected from multiple sources between 1986 and 2020 were used. Pre-treatment reverse transcriptase inhibitor (RTI), PI and integrase inhibitor (INSTI) mutations were assessed using the Stanford HIVDR database. Phylogenetic methods were used to determine and date transmission clusters. Results Of 3567 sequences analysed, 550 (15.4%, 95% CI: 14.2–16.6) had at least one pre-treatment HIVDR mutation, which was most prevalent amongst children (41.3%), followed by PWID (31.0%), MSM (19.9%), FSWs (15.1%) and HETs (13.9%). Overall, pre-treatment HIVDR increased consistently, from 6.9% (before 2005) to 24.2% (2016–20). Among HETs, pre-treatment HIVDR increased from 6.6% (before 2005) to 20.2% (2011–15), but dropped to 6.5% (2016–20). Additionally, 32 clusters with shared pre-treatment HIVDR mutations were identified. The majority of clusters had R0 ≥ 1.0, indicating ongoing transmissions. The largest was a K103N cluster involving 16 MSM sequences sampled between 2010 and 2017, with an estimated time to the most recent common ancestor (tMRCA) of 2005 [95% higher posterior density (HPD), 2000–08], indicating propagation over 12 years. Conclusions Compared to HETs, children and key populations had higher levels of pre-treatment HIVDR. Introduction of INSTIs after 2017 may have abrogated the increase in pre-treatment RTI mutations, albeit in the HET population only. Taken together, our findings underscore the need for targeted efforts towards equitable access to ART for children and key populations in Kenya.