Background: Studies suggest that mitochondrial DNA (mtDNA) haplogroups are associated with antiretroviral therapy (ART)-related metabolic complications and distal sensory polyneuropathy (DSP), but there have been few studies in persons of African descent. We explored such associations in South African adults. Methods: Clinical and laboratory data and DNA specimens from a cross-sectional study were used. Sequencing and Phylotree determined African mtDNA subhaplogroups. Wilcoxon and regression analyses determined associations between mtDNA subhaplogroups and ART-related complications. Results: The 171 participants represented 6 major haplogroups: L0 (n=78), L1 (n=3), L2 (n=30), L3 (n=53), L4 (n=1) and L5 (n=6). Analyses were restricted to 161 participants representing L0, L2 and L3: 78% female; median age 36 years. All had been exposed to thymidine analogues, 42% were on lopinavir/ritonavir (lopinavir/r) and 58% were on either efavirenz or nevirapine. Median (IQR) ART duration was 22 (14-36) months. Median fasting triglycerides were 1.60 (1.13-1.75) and 1.04 (0.83-1.45) mmol/L among L3e1 (n=22) and other subhaplogroups, respectively (p=0.003). Subhaplogroup L3e1 (adjusted OR (aOR) 3.15 [95% CI: 1.11-8.96]; p=0.03) and exposure to lopinavir/r (aOR 2.98 [95% CI: 1.02-8.96]; p=0.05) were independently associated with hypertriglyceridaemia, after adjusting for age, sex and ART duration. There were no significant associations between mtDNA haplogroups and cholesterol, dysglycaemia, hyperlactataemia, or lipoatrophy, or DSP. Conclusions: Subhaplogroup L3e1 and lopinavir/r exposure were independently associated with hypertriglyceridaemia in black South Africans on ART. This is the first report to link an African mtDNA variant with hypertriglyceridemia. If replicated, these findings may provide new insights into host factors affecting metabolic complications.