Introduction: HIV is a retrovirus that replicates slowly and is responsible for acquired immunodeficiency syndrome (AIDS) in humans. Immune system is weakened ultimately making infected individuals more vulnerable to numerous secondary infections. According to an estimate, HIV has infected more than seventy million people since 1981 and is responsible for the death of 35 million people so far. By the end of year 2016, 36.7 million population were found to be living with HIV worldwide. Pakistan, a developing nation of 200 million inhabitants, is witnessing an increase in the number of HIV infected individuals. The improved use of antiretroviral therapy (ART) has reduced the morbidity and mortality linked with HIV, however, at the cost of the emergence of HIV drug resistance strains (HIVDR). No significant data exist about the epidemiology of HIV-1 genotypes and the drug resistance mutations. Objectives: To determine the molecular epidemiology of HIV-1 and its correlation with antiretroviral drug resistance among AIDS patients. Study design: Cross-sectional, prospective multi-centre study. Duration: January 2015 – June 2018. Setting: Department of Blood Transfusion Services, Shaheed Zulfiqar Ali Bhutto Medical University, Islamabad; Department of Pathology, Jinnah Postgraduate Medical Complex, Karachi; and Department of Biotechnology and Molecular Biology, International Islamic University, Islamabad. Methods: A total of 410 HIV-positive patients (both on treatment and treatment naïve) were recruited in the study. From the Voluntary Counselling and Treatment Centre (VCTC), Jinnah Postgraduate Medical Centre (JPMC), Karachi, blood samples were collected from 298 HIV/AIDS patients on antiretroviral therapy (ART). For the treatment of naïve individuals, a community based survey on 387 high risk group individuals was conducted in different cities yielding 37 HIV positive samples. In addition, 54,877 blood donors were screened for HIV-1&2 at the Department of Blood Transfusion Services, SZAB Medical University, of which, 75 were found reactive. HIV screening was performed by rapid point of care HIV screening device (AlereDetermineTM HIV-1/2, Alere North America Inc. USA). All samples were confirmed by the chemiluminescence immunoassay using fully automated Abbott Architect i2000SR system. The samples tested positive were re-tested using Abbott’s CLIA system. Using standard questionnaire, the study subjects were also interviewed regarding their living conditions, daily routines, travel history and sexual behaviour. Using standard methods, viral RNA of HIV was extracted from the blood specimens of positive patients, and was converted to cDNA. HIV cDNA of all positive patients was then analysed for the presence of various HIV genotypes (types and sub-types) by employing subtype-specific primers in a nested PCR (polymerase chain reaction). Sanger sequencing standard protocols was followed to detect the mutations in the genes related to drug resistance in HIV. All the data and samples were kept confidential and anonymous. HIV analyses was performed according to the conditions of “5-Cs”: comprising of informed consent, be confidential, involve counselling, deliver correct test results and connections to prevention, treatment and care services. Informed written consent was received from all study subjects participating in this study. Results: A total of 387 subjects from selected high risk groups (HRGs) agreed to provide blood sample. Out of 387, a total of 149 subjects tested positive for syphilis (38.5%), whereas 37 tested positive for HIV (9.6%). Syphilis co-infection was found in 22 of the HIV infected subjects (59.5%; odd ratio 2.53; p=0.008). The HIV screening of 54,877 blood donors initially yielded 77 reactive cases. A repeat testing showed 0.13% (n=75) positive cases (Fig 4.2), with 95% confidence intervals 0.0014 (0.0011 – 0.0018). No female donor was reactive for HIV. From the genotypic analysis of 410 HIV positive individuals, the predominant HIV-1 subtype was A (n=376) (91.7%) followed by subtype B (n=34) (8.3%). The results of reverse transcriptase region analysis for resistance mutations exhibited that 89% of the sequences do not have major and minor mutations. The percentage of sequences showing a major mutation was 11%. The major mutation was Y115F, where the patient sample is having Tyrosine (Tyr) at position 115, while the normal individual have Phenylalanine (Phe). The results of PR region analysis showed no major mutations. On the other hand, minor mutations were exhibited by six sequences. Two of the mutations were categorized as L10V, and the remaining four included A71AV, L10FL, G48GR and L10I. Conclusions: The present study has provided a complete baseline data on the molecular and genetic characterization of HIV/AIDS epidemic in Pakistan. Further studies of antiretroviral drug resistance mutations would help in streamlining resistance pattern and subsequent alternate therapies.