Shandong province has been providing antiretroviral therapy (ART) to eligible HIV/AIDS patients since 2003 using first-line regimens. We conducted a cross-sectional study to assess virological response and resistance development from ART patients. Between 2006 and 2008, blood was collected from 143 ART patients. Viral load (VL) was determined with a detection limit of 50 copies/ml; those with detectable VL were genotyped with dried plasma spots using a broadly sensitive genotyping assay. Resistance mutations were identified using the Stanford HIV drug resistance database. Of the 143 patients, 72% [95% confidence interval (CI): 65.9-78.2] suppressed their VL to <50 copies/ml. Genotyping analysis of the remaining 40 patients revealed that 21 (53%, CI: 37.0-68.0) harbored one or more mutations. The most common mutations were thymidine-analog mutations (22.5%) and M184V (10%) to nucleoside reverse transcriptase inhibitors (NRTIs), and V106I/A /M (17.5%), Y181C (15%), and H221Y (12.5%) to non-NRTIs (NNRTIs); 13 patients had mutations to both NRTIs and NNRTIs. Patients with VL >1000 copies/ml appear to harbor more mutations than those with VL between 50 and 1000 (62.1% vs. 27.3%, p>0.05). Resistance mutations were intensified among 10 patients for whom two sequential specimens were obtained and accumulation of resistance mutations predicted compromised treatment outcomes and future drug selections. This study provides a snapshot of the virological responses and resistance profiles for patients on first-line regimens, indicating that patient monitoring is a critical component in preventing the accumulation of resistance mutations among patients failing their regimens and thus maintaining the effectiveness of the first-line regimens.