Cortisol is a hormone released by the adrenal cortex in stressful situations. Dogs housed in shelters are known to have higher cortisol levels than those residing in permanent homes. This study was conducted to investigate various aspects of shelter environment, backgrounds in shelter entry, and behavioral effects on cortisol levels in dogs from five Pennsylvania and Maryland shelters. Saliva samples from 105 dogs were analyzed using a sensitive immunoassay for cortisol. Several factors were investigated: shelter conditions, background of dogs (i.e. strays and owner surrenders), breed, and time in shelter. The interaction of behavioral indices and cortisol was also investigated. Shelter conditions significantly affect cortisol levels of dogs when adjusted for their behavioral index. Behavioral index shows a significant negative correlation with cortisol when adjusted for background. Background affects cortisol levels; strays have significantly higher cortisol levels than owner surrenders. Breed and length of time in shelter had no effect on cortisol levels. These results can be used to improve shelter conditions to avoid causing further stress to dogs.