Abstract Background In the perspective of ART-free HIV remission, vertically infected children treated with suppressive ART from early infancy represent an optimal population model to better understand the genetic complexity of the reservoir. Objectives To evaluate the proportion of defective viral population and the genotypic resistance patterns in cell-associated HIV DNA. Methods In a cohort including 93 ART-treated vertically HIV-infected (VHIV) children in Mali with plasma HIV-1 RNA ≤50 copies/mL for at least 6 months, we studied total HIV DNA, percentage of defective genomes and resistance by reverse transcriptase and protease bulk sequencing from whole blood in dried blood spots. Results Children had a median age of 9.9 years at the time of inclusion (IQR = 7.6–13.4) and 3.3 years (IQR = 2–7) at ART initiation; median ART duration was 5.5 years (IQR = 3.7–7.3). The median level of total HIV DNA was 470 copies/106 cells with one patient presenting undetectable HIV DNA (<66 copies/106 cells). We observed the presence of at least one stop codon in viruses from 34 patients (37%). The presence of stop codons was not correlated with the level of HIV DNA or duration of ART. We showed a high prevalence of HIV-1 resistance in DNA with 26% of children harbouring virus resistant to at least one NRTI and 40% to at least one NNRTI. Conclusions While these VHIV children were successfully treated for a long time, they showed high prevalence of resistance in HIV DNA and a moderate defective HIV reservoir.