Dogs with atopic dermatitis (AD) often exhibit behaviors such as scratching and rubbing. This discomfort may cause stress in affected dogs. Thus, we investigated the association between stress levels in companion dogs with and without AD using hair cortisol concentration (HCC). In total, 202 dogs were involved in this study, including bichon frise (24 AD, 38 non-AD, 5.3 ± 1.8 kg), maltese (17 AD, 51 non-AD, 3.4 ± 0.8 kg), and poodle (14 AD, 58 non-AD, 4.4 ± 1.4 kg). Hair samples were collected by the owners once from the dog's neck, close to the skin, using scissors from 2019 to 2023 in Korea. The HCC was determined using a commercial enzyme immunoassay kit. Based on these results, the HCC in bichon frise and poodle with AD were higher ( < 0.05) than those without AD. No difference ( > 0.05) in HCC was observed in maltese with AD and without AD. Pooled data showed a higher HCC ( < 0.05) in AD dogs compared to without AD dogs. The HCC in female bichon frise and female maltese with AD were higher ( < 0.05) than those without AD. Conversely, no differences ( > 0.05) in HCC were observed between AD and non-AD in male bichon frise and male maltese dogs. Higher ( < 0.05) HCC were observed between the AD and non-AD in female and male poodles. In conclusion, the AD condition in dogs can act as a stressor and analysis of HCC can help to better monitor the chronic stress level of dogs affected by AD.