We recently constructed two rhesus macaque-tropic human immunodeficiency virus type 1 (HIV-1rmt) clones with CXCR4 or CCR5 tropism, but a CCR5-tropic HIV-1rmt clone grew more poorly than a CXCR4-topic clone. It has been demonstrated that interaction between viral Gag-matrix (MA) and Env-gp41 cytoplasmic tail is important for virion-incorporation of Env. Concordantly, Gag-MA mutations (62QR and 66SR) that rescue defects in virion-incorporation of Env/viral replication were reported. In this study, we analyzed effects of these Gag-MA mutations on R5-tropic HIV-1rmt replication potentials. While introduction of 62QR into three HIV-1rmt clones tested reduced their multi-cycle replication ability in rhesus lymphocytes or abolish single-cycle infectivity for luciferase reporter cells, three R5-tropic HIV-1rmt clones carrying 66SR exhibited similar growth kinetics to those of their parental clones. One such clone, 66SR+5gtu, appeared to induce stronger cytopathic effects than parental clone 5gtu. We therefore investigated effects of 66SR mutation on viral replication in more detail. Single-cycle infectivity of 66SR+5gtu was enhanced relative to that of 5gtu, but 66SR+5gtu virion production was significantly decreased compared to the 5gtu level. Gag-MA 66SR mutation may be useful to improve growth potentials of the R5-tropic HIV-1rmt clones.