A simple and sensitive diagnosis method is needed to identify HIV infection in sera of untreated, treated, and drug-resistant patients. The purpose of this study is to determine whether heat shock proteins (Hsp)-27 and -20 and HP91 peptide along with HIV-1 polypeptides can serve as potential biomarkers to distinguish HIV infection in untreated, treated, and drug-resistant individuals compared to HIV-negative subjects. Methods: At first, human sera were obtained from 141 participants, including 20 naïve HIV-infected, 71 treated, 30 drugresistant, 20 HIV-negative (healthy/control) individuals. The recombinant Hsp27, Hsp20, and five designed HIV-1 polypeptides were expressed in Escherichia coli and purified by affinity chromatography under denaturing or native conditions. Finally, the antibodies against these antigens were quantified in sera using ELISA. Results: Our data showed that HIV-infected patients significantly displayed higher serum levels of anti-Hsp27, anti-HP91, and anti-Nef-Tat-Gp160-P24, anti-Nef-Vpr-Gp160-P24, anti-Nef-Vif-Gp160-P24, anti-Nef-Vpu-Gp160-P24, and anti-Nef-Rev-Gp160-P24 polypeptide antibodies than healthy groups (p < 0.05), but not for anti-Hsp20. Moreover, the serum levels of antibodies against Hsp27, Hsp20, HP91, and HIV-1 polypeptides were not statistically significant between different groups of patients (p > 0.05). Conclusion: The levels of anti-Hsp27 and anti-HP91 antibodies in serum increased in HIV-1 seropositive subjects along with antibodies against five HIV-1 polypeptides suggesting their potential value as a diagnostic marker for HIV-1 infections.