ABSTRACTChronic kidney disease (CKD) and obesity are major chronic diseases in the United States. Although obesity is a risk factor for CKD, little is known about how obesity contributes to CKD. Due to their role as intercellular communicators, extracellular vesicles (EVs) may be a factor connecting obesity and CKD. Circulating cell‐free mitochondrial DNA (ccf‐mtDNA), a damage‐associated molecular pattern molecule associated with inflammation, is associated with renal disease and may be encapsulated within EVs. In this longitudinal study, we isolated plasma EVs and analysed EV mtDNA levels in a cohort of African American and White obese middle‐aged individuals who at visit 1 did not have CKD but developed CKD by visit 2 (n = 19; CKD group) and matched this group to controls who did not develop CKD by visit 2 (n = 56; control group). In our cross‐sectional analyses at visit 1, we found significant interactions for EV mtDNA levels between race and CKD status, poverty status and CKD status, and sex and CKD status. EV mtDNA levels were significantly lower in participants within the African haplogroup who developed CKD compared to participants within the European haplogroup who developed CKD and the African haplogroup control group. In our longitudinal analyses using data from both visit 1 and visit 2, individuals who developed CKD had lower EV mtDNA levels. Stratification by haplogroup showed that among participants within the African haplogroup, those who developed CKD had significantly lower EV mtDNA levels than those in the control group. In conclusion, EV mtDNA levels were lower in individuals who develop CKD. Our findings demonstrate that CKD status and mtDNA haplogroup influence EV cargo in obese individuals.