ABSTRACT Abacavir is frequently used in antiretroviral combination therapies as a potent nucleoside reverse transcriptase inhibitor (NRTI). Four mutations are selected for by abacavir in vitro and in vivo: K65R, L74V, Y115F, and M184V. Abacavir resistance has also been observed in NRTI multidrug-resistant samples. Furthermore, abacavir resistance has been described in the context of zidovudine resistance. To evaluate the genetic basis of abacavir resistance, the viral genotype and phenotypic resistance were analyzed for 307 patient samples. Low- and high-level resistances were defined as 2.5- to 5.5-fold- and >5.5-fold-reduced susceptibility, respectively. If all samples with abacavir-selected and NRTI multidrug resistance-associated mutations were scored as resistant, 27.6% of the samples were misclassified, mainly due to samples falsely scored as susceptible. Therefore, the relative frequencies of other mutations were evaluated. Mutations at codons 44 and 118 were rarely detected in abacavir-susceptible samples but were overrepresented in resistant samples. Site-directed mutagenesis of E44D, V118I, and M184V resulted in low-level resistance for the double mutant 44/184 and the triple mutant. Low-level abacavir resistance was also detected for a viral clone carrying zidovudine mutations only. Additional insertion of M184V into the zidovudine background doubled the resistance, whereas 44/118 did not lead to a further increase. Incorporating combinations of zidovudine mutations and M184V into the scoring system markedly reduced the number of misclassified samples, whereas 44/118 did not improve the prediction. In conclusion, the combination of M184V with zidovudine mutations gives rise to high-level abacavir resistance, which may be clinically relevant. Thus, options for useful sequential combinations of NRTI are limited.