No previous study has explored the relationship between cytology and the frequency of behaviours associated with anal sac disease (ASD). The goals of the study were: (i) to compare the cytological findings between anal sac secretions from normal dogs with no history of ASD to those with non-neoplastic ASD; (ii) to determine whether anal sac cytological findings can be used to differentiate between normal dogs and dogs with ASD; (iii) to explore the correlation of anal sac cytology and behaviour between normal dogs and dogs with ASD; and (iv) to describe behaviours typical of ASD as reported by owners. Thirty dogs were selected for this study, based on their behavioural history as detailed in a questionnaire completed by their owners. Of the thirty dogs, ten were considered normal insofar as they had no history of ASD clinical signs. The remaining 20 dogs were characterized as having ASD, with a chronic history of perianal pruritus, but no other pruritus. All dogs had their anal sacs manually expressed, and the discharge was examined microscopically in a blinded manner. A total of 171 oil immersion fields (OIFs) were examined from normal dogs and 333 OIFs from dogs with ASD. The behavioural results for dogs with ASD revealed that scooting recurred with a median frequency of 3 weeks post-anal sac expression. There were no clinically statistically significant cytological differences between normal dogs and those with ASD, thereby leading to the conclusion that cytology is an ineffective tool for diagnosing ASD.