Shelter dog populations in the United States are poorly quantified and characterized, but may be effective targets for measuring the occurrence of select diseases affecting animal and human health. Dogs in this population may have increased risk for disease due to intrinsic and extrinsic risk factors. Accurate estimates of disease in this population require sound sampling strategies within a comprehensive sampling frame. Knowledge of the prevalence of disease in the Mississippi shelter dog population is important for diagnostic test interpretation, shelter allocation of resources, and public health risk assessment. A serum bank provides a valuable resource to investigate both zoonotic diseases in which dogs are the primary reservoir, such as canine brucellosis, and for diseases where dogs may be effective sentinels for exposure risk, such as American trypanosomiasis. Implications of this research extend beyond Mississippi through the frequent movement of shelter dogs to adoption centers across the United States.