Background: Until 2019, first-line antiretroviral therapy (ART) in Southern Africa consisted of one non-nucleoside reverse transcriptase inhibitor (NNRTI) and two nucleoside reverse transcriptase inhibitors (NRTI). As a response to the increasing level of NNRTI resistance, these drugs are being replaced by dolutegravir (DTG), an integrase inhibitor with a high barrier to resistance. Patients failing an NNRTI-based regimen might therefore start DTG-based therapy with preexisting NRTI resistance, potentially jeopardizing the long-term success of DTG-based ART. We performed a systematic review and meta-analysis to quantify the prevalence of NRTI drug resistance mutations (DRMs) in patients failing NNRTI-based ART in Southern Africa. Methods: We searched several bibliographic databases, including Embase and Medline, from inception to May 2019 to identify studies reporting NRTI DRMs observed among adult HIV-positive patients experiencing virological failure on first-line NNRTI-based regimens in countries of Southern Africa. After screening titles and abstracts, two independent reviewers assessed full manuscripts of potentially eligible studies and extracted data. We developed a hierarchical logistic meta-regression model to synthesize the effect of different ART regimen on the emergence of NRTI and NNRTI DRMs across studies, accounting for ART duration and study-specific effects. Analyses were performed in a Bayesian framework using the rstan package in R.Results: Of 7,579 studies, 3,247 were duplicates and 4,135 were excluded after initial screening. After assessing 194 full-texts, we included 15 studies with 17 study samples and 2,432 individuals from South Africa (13 studies), Mozambique (1), Botswana (1), Lesotho (1) and Zambia (1). We analyzed the dynamics of nine NRTI DRMs by ART regimen. Baseline levels of DRMs were low, ranging from 0.2% to 7.8%. The use of emtricitabine/lamivudine was associated with development of high levels of the M184V/I mutation (1.2% at baseline vs. 64% after 3 years on treatment). When emtricitabine/lamivudine was combined with tenofovir disoproxil fumarate, a substantial increase in the K65R mutation (0.8% at baseline vs. 69.5% after 3 years) was observed. We also analyzed the dynamics of seven NNRTI DRMs after 3 years. With a prevalence of 45.6% after 3 years of efavirenz, K103 was the most prevalent NNRTI resistance mutation, followed by V106 (35.5% after 3 years of efavirenz) and Y181 (14.7% after 3 years of nevirapine).Interpretation: In patients failing first-line ART in Southern Africa, the prevalence of NRTI DRM is high, suggesting that a substantial proportion of patients failing NNRTI-based regimen will switch to DTG-based regimen with non-working NRTIs. This could potentially impair the long-term efficacy of DTG-introduction in Southern Africa.