In South Vietnam, live bird markets (LBMs) are key in the value chain of poultry products and spread of avian influenza virus (AIV) although they may not be the sole factor to determine avian influenza (AI) prevalence in the southern part. Therefore, a risk analysis of AIV spread was conducted by including all possible value chain factors. A cross-sectional study was performed in backyard farms, high-biosecurity farms (bio-farms), LBMs, and poultry delivery stations (PDSs) in the four districts of Vinh Long Province in December 2016 and August 2017. A total of 3 597 swab samples were collected from individual poultry at 101 backyard farms, 50 bio-farms, 58 sellers in LBMs, and 17 traders in PDSs and then investigated for AIV isolation. Concurrently, information related to participants and birds was collected and used to identify the fixed and random effects of factors in AIV infection. A total of 274 birds were positive for virus isolation, with a prevalence of 7.6% (95% confidence interval [CI]: 6.8–8.5) at the individual poultry level, and the adjusted prevalence based on the sampling weight was 7.9% (95% CI: 7.6–8.2). The significantly higher prevalence in PDSs (20.7%) and LBMs (14.2%) compared to backyard farms (3.0%) and bio-farms (0.6%) suggested that PDSs are another hot spot for AIV circulation. The high diversity in the seller and trader population characteristics was revealed using multiple-correspondence analysis to analyze the participants’ demographic factors in LBM and PDS. The mixed-effect logistic regression model revealed that keeping duck at the sampling time and the owner’s older age should be risk factors of AIV infection in PDS. Therefore, functional AI control efforts to monitor the PDS system should be emphasized to minimize AIV circulation risk in Vietnam.