The Southeast accounted for most HIV diagnoses (52%) in the United States in 2015. Primary care providers (PCPs) play a vital role in HIV prevention for at-risk persons and treatment of persons living with HIV. We studied HIV-related training, knowledge, and clinical practices among PCPs in the Southeast to address knowledge gaps to inform HIV prevention strategies. Between April and August 2017, we conducted an on-line survey of a representative sample of PCPs in six Southeast jurisdictions with high rates of HIV diagnoses (Atlanta; Baltimore; Baton Rouge; District of Columbia; Miami; New Orleans). We defined HIV-related training as self-reported completion of any certified HIV/STD course or continuing education in past 24 months (prior to survey completion). We assessed associations between training and HIV testing practices, familiarity with nonoccupational post-exposure prophylaxis (nPEP) and pre-exposure prophylaxis (PrEP), and ever prescribing nPEP or PrEP. There were 820 participants after fielding 4595 surveys (29.6% adjusted response rate). In weighted analyses, 36.3% reported HIV-related training. Using adjusted prevalence ratio (aPR) and confidence intervals (CI), we found that PCPs with HIV-related training (compared to those with no training) were more likely to be familiar with nPEP (aPR = 1.32, 95% CI 1.05, 1.67) and PrEP (aPR = 1.67, 95% CI 1.19, 2.38); and to have ever prescribed PrEP to patients (aPR = 1.75, 95% CI 1.10, 2.78). Increased HIV-related trainings among PCPs in high HIV prevalence Southeast jurisdictions may be warranted. Strengthening nPEP and PrEP familiarity among PCPs in Southeast may advance national HIV prevention goals.