The HIV-1 integrase, responsible for the chromosomal integration of the newly synthesized double-stranded viral DNA into the host genomic DNA, represents a new and important target of potential clinical relevance. For instance, two integrase inhibitors, raltegravir and elvitegravir, have been shown to be promising in clinical trials, and the first has been recently made available for clinical practice. As is the case for other antiviral drugs, drug resistance to integrase inhibitors occurs both in vitro and/or in vivo through the selection of mutations within the HIV genome. Indeed, many integrase mutations have already been associated with resistance to all the different integrase inhibitors tested in in vitro and/or in vivo studies. Among them, about 40 substitutions have been specifically associated with the development of resistance to raltegravir and/or elvitegravir; some of them were also found in vivo in patients failing such integrase inhibitors. The relevance of integrase mutations in clinical practice has yet to be defined, in light of the lack of long-term follow-up of treated patients and the limited data about the prevalence of integrase inhibitor-associated mutations in integrase inhibitor-naive patients (either untreated, or treated with antiretrovirals not containing integrase inhibitors). Therefore, by structural analysis elaboration and literature discussion, the aim of this review is to characterize the conserved residues and regions of HIV-1 integrase and the prevalence of mutations associated with integrase inhibitor resistance, by matching data originated from a well-defined cohort of HIV-1 B subtype-infected individuals (untreated and antiretroviral-treated) and data originated from the public Los Alamos Database available in the literature (all patients integrase inhibitor-naive by definition). In integrase inhibitor-naive patients, 180 out of 288 HIV-1 integrase residues (62.5%) are conserved (< 1% variability). Residues involved in protein stability, multimerization, DNA binding, catalytic activity, and in the binding with the human cellular cofactor LEDGF/p75 are fully conserved. Some of these residues clustered into large defined regions of consecutive invariant amino acids, suggesting that consecutive residues in specific structural domains are required for the correct performance of HIV-1 integrase functions. All primary signature mutations emerging in patients failing raltegravir (Y143R, Q148H/K/R, N155H) or elvitegravir (T66I, E92Q, S147G, Q148H/K/R, N155H), as well as secondary mutations (H51Y, T66A/K, E138K, G140S/A/C, Y143C/H, K160N, R166S, E170A, S230R, D232N, R263K) were completely absent or highly infrequent (< 0.5%) in integrase inhibitor-naive patients, either infected with HIV-1 B subtype (drug-naive or antiretroviral-treated), or non-B subtypes/group N and O. Differently, other mutations (L74M, T97A, S119G/R, V151I, K156N, E157Q, G163K/R, V165I, I203M, T206S, S230N) occurred as natural polymorphisms with a different prevalence according to different HIV-1 subtype/circulating recombinant form/group. In conclusion, the HIV-1 integrase in vivo is an enzyme requiring the full preservation of almost two-thirds of its amino acids in the absence of specific integrase inhibitor pressure. Primary mutations associated with resistance to integrase inhibitors clinically relevant today are absent or highly infrequent in integrase inhibitor-naive patients. The characterization of the highly conserved residues (involved in protein stability, multimerization, DNA binding, catalytic activity, LEDGF binding, and some with still poorly understood function) could help in the rational design of new HIV-1 inhibitors with alternative mechanisms of action and more favorable resistance profiles.