ABSTRACT Human immunodeficiency viruses in 321 samples from tenofovir-naïve patients were retrospectively evaluated for resistance to this nucleotide analogue. All virus strains with insertions between amino acids 67 and 70 of the reverse transcriptase ( n = 6) were highly resistant. Virus strains with the Q151M mutation were divided into susceptible ( n = 12) and highly resistant ( n = 8) viruses. This difference was due to the absence or presence of the K65R mutation, which was confirmed by site-directed mutagenesis. Viral clones with various combinations of the mutations M41L, K70R, L210W, and T215F or T215Y were analyzed for cross-resistance induced by thymidine analogue mutations (TAMs). The levels of increased resistance induced by single, double, and triple mutations at the indicated positions could be ranked as follows: for mutants with single mutations, mutations at positions 41 > 215 > 70; for mutants with double mutations, mutations at positions 41 and 215 > 70 and 215 = 210 and 215 > 41 and 70; for mutants with triple mutations, mutations at positions 41, 210, and 215 > 41, 70, and 215. Viral clones with M184V or M184I exhibited slightly increased susceptibilities to tenofovir (0.7-fold). Almost all clones with TAM-induced resistance were resensitized when M184V was present ( P < 0.001). Among the viruses in the clinical samples, the rate of tenofovir resistance significantly increased with the number of TAMs both in the samples with 184M and in those with 184V ( P = 0.005 and P = 0.003, respectively). A resensitizing effect of M184V was confirmed for all samples exhibiting at least one TAM ( P = 0.03). However, accumulation of at least two TAMs resulted in more than 2.0-fold reduced susceptibility to tenofovir, irrespective of the presence of M184V. Decision tree building, a classical machine learning technique, was used to generate models for the interpretation of mutations with respect to tenofovir resistance. The application of previously proposed cutoffs for a reduced response to therapy and treatment failure demonstrated the central roles of positions 215 and 65 for 1.5- and 4.0-fold reduced susceptibilities, respectively. Thus, clinically relevant resistance may be conferred by the accumulation of TAMs, and the resensitizing effect of M184V should be considered only minor.