Lenacapavir (LEN; GS-6207) is a potent first-in-class inhibitor of HIV-1 capsid with long-acting properties and the potential for subcutaneous dosing every 3 months or longer. In the clinic, a single subcutaneous LEN injection (20 mg to 750 mg) in people with HIV (PWH) induced a strong antiviral response, with a >2.3 mean log decrease in HIV-1 RNA at day 10. HIV-1 Gag mutations near protease (PR) cleavage sites have emerged with the use of protease inhibitors (PIs). Here, we have characterized the activity of LEN in mutants with Gag cleavage site mutations (GCSMs) and mutants resistant to other drug classes. HIV mutations were inserted into the pXXLAI clone, and the resulting mutants ( = 70) were evaluated using a 5-day antiviral assay. LEN EC fold change versus the wild type ranged from 0.4 to 1.9 in these mutants, similar to that for the control drug. In contrast, reduced susceptibility to PIs and maturation inhibitors (MIs) was observed. Testing of isolates with resistance against the 4 main classes of drugs ( = 40) indicated wild-type susceptibility to LEN (fold change ranging from 0.3 to 1.1), while reduced susceptibility was observed for control drugs. HIV GCSMs did not impact the activity of LEN, while some conferred resistance to MIs and PIs. Similarly, LEN activity was not affected by naturally occurring variations in HIV Gag, in contrast to the reduced susceptibility observed for MIs. Finally, the activity of LEN was not affected by the presence of resistance mutations to the 4 main antiretroviral (ARV) drug classes. These data support the evaluation of LEN in PWH with multiclass resistance.