Abstract Objectives To describe plasma residual HIV viraemia, cellular HIV reservoir size, blood plasma drug concentrations and their male genital tract penetration during the maintenance dual therapy dolutegravir\,+\,lamivudine. Patients and methods ANRS167 LAMIDOL enrolled 104 virologically suppressed patients to switch to dolutegravir\,+\,lamivudine. In this pharmacovirological substudy, ultrasensitive plasma viral load (USpVL) and plasma drug concentrations were measured at Day 0 (D0), Week 24 (W24) and W48 of dolutegravir\,+\,lamivudine, and HIV-DNA was measured at W-8 and W48. Semen samples were collected at D0 and W24 from 18 participants. Total and unbound blood and seminal plasma drug concentrations were measured using UPLC\textendash MS/MS. Results Median HIV-DNA was 2.5\,log10\,copies/106 PBMC (IQR\,=\,2.2\textendash 3.0, n\,=\,100) at W-8 and 2.4\,log10\,copies/106 PBMC (IQR\,=\,2.1\textendash 2.9, n\,=\,100) at W48 (P\,=\,0.17). The proportion of patients with undetected USpVL was 38% (n\,=\,98), 43% (n\,=\,98) and 49% (n\,=\,97) at D0, W24 and W48, respectively (P\,=\,0.08). Total and unbound plasma dolutegravir concentrations were stable between timepoints (P\,=\,0.13) and all total plasma dolutegravir concentrations except one were adequate. Median free fraction of dolutegravir in plasma was 0.21%. Median blood plasma and seminal plasma concentrations of total dolutegravir at 24\,h were 1812\,ng/mL and 206\,ng/mL, respectively. Median seminal plasma/blood plasma total concentration ratios were 11.6% and 2478% for dolutegravir and lamivudine, respectively. HIV-RNA (365 to 475\,copies/mL) was detected in seminal plasma of one patient at D0 (5.9%) and of two patients at W24 (11.8%). Conclusions These findings add further important information regarding the effectiveness of dolutegravir\,+\,lamivudine maintenance dual therapy in terms of plasma residual viraemia, cellular reservoir size and drug penetration in the male genital tract.