The mission of the Region IV (R-IV) Public Health Training Center (PHTC), headquartered at the Rollins School of Public Health at Emory University in Atlanta, GA, is to develop and implement programming to train and educate public health professionals in U.S. Department of Health and Human Services Region IV. To identify public health workforce development needs, the R-IV PHTC created a systematic process that included the implementation of a variety of strategies, to gain insights from each state within the diverse region. Conducting regular needs assessments is an integral step to ensure trainings are relevant and meet the needs of public health professionals. To this end, the PHTC employed a mixed methods approach to gather information on both competency-based and non–competency-based training needs, as well as training needs within R-IV’s content focus area of infectious disease. In R-IV there is great variability between the structures of the state and local health departments (e.g., some centralized, some decentralized), each of which faces different funding challenges and works with different service delivery models and regulatory authorities. Moreover, states have diverse populations (e.g., races, urban/rural, migrant/refugees, tribal, Appalachian) and face a wide range of public health priority concerns. Health departments were found to be at different stages of readiness to undertake a training needs assessment due to a number of issues, including their stage of pursuing Public Health Accreditation Board accreditation and recent participation in other needs assessment efforts. The R-IV PHTC approach to assessing training needs within this challenging environment is described.