Some mutations in the connection subdomain of the polymerase domain and in the RNase H domain of HIV-1 reverse transcriptase (RT) have been shown to contribute to resistance to RT inhibitors. However, the clinical relevance of such mutations is not well understood. To address this point we determined the prevalence of such mutations in a cohort of antiretroviral treatment-naïve patients (n=123) and assessed whether these substitutions are associated with drug resistance in vitro and in vivo. We report here significant differences in the prevalence of substitutions among subtype B, and non-subtype B HIV isolates. Specifically, the E312Q, G333E, G335D, V365I, A371V and A376S substitutions were present in 2–6% of subtype B, whereas the G335D and A371V substitutions were commonly observed in 69 and 75% of non-B HIV-1 isolates. We observed a significant decline in the viral loads of patients that were infected with HIV-1 carrying these substitutions and were subsequently treated with triple drug regimens, even in the case where zidovudine (AZT) was included in such regimens. We show here that generally, such single substitutions at the connection subdomain or RNase H domain have no influence on drug susceptibility in vitro by themselves. Instead, they generally enhance AZT resistance in the presence of excision-enhancing mutations (EEMs, also known as thymidine analogue-associated mutations, TAMs). However, N348I, A376S and Q509L did confer varying amounts of nevirapine resistance by themselves, even in the absence of EEMs. Therefore, our cohort establishes that several connection subdomain and RNase H domain substitutions typically act as pre-therapy polymorphisms.