During its spread among humans, HIV-1 has developed an extraordinary degree of genetic diversity. The pol region encoding for viral enzymes such as the reverse transcriptase and the protease, and the env region encoding for the viral glycoprotein gp41 are subjected not only to natural variation, but also to the selection pressure imposed by the pharmacologic treatment. Under these conditions in HIV-1 infected people, the virus is able to escape from antiviral drugs by accumulating mutations, either alone or in clusters. The patterns of mutations accumulated by HIV-1 under drug pressure are quite variable, depending on the backbone of virus strains, the level and type of pharmacologic pressure, and the length of therapy. To date, a high number of mutations in protease, reverse transcriptase, and gp41 have been associated with reduced susceptibility to the antiretroviral drugs currently available. However, a number of studies continuously highlight the existence of additional mutations beyond those currently known to be involved in the development of drug resistance in vivo. Most of these so-called "novel" mutations are involved in agonistic correlations with the classical drug resistance mutations on divergent evolutionary pathways, and are associated with an increased resistance to specific drugs. At the same time, the presence of some novel mutations at therapeutic failure has also been significantly associated with an increase of viremia, thus suggesting that they may also play a compensatory role leading to improved viral replication. Interestingly, some natural polymorphisms in drug-naive patients have been significantly associated with the development of drug resistance mutations at failure, thus suggesting their ability to decrease the genetic barrier to the development of drug resistance. In contrast, other novel mutations are negatively associated with specific antiviral treatment, showing negative interactions with relevant drug resistance mutations, and are associated with increased susceptibility to specific drugs. This article reviews the importance of recognition and the clinical relevance of novel mutations involved in resistance to the currently used antiretroviral drugs, discussing in particular the role of novel drug resistance mutations in the reverse transcriptase enzyme. Such novel mutations should be considered for improved prediction of clinical response to antiretroviral drugs and for assessing the efficacy of next-generation drugs.