Mammary gland tumors are by far the most commonly found tumors in domestic dogs. Effective therapeutic procedures with prompt accurate diagnoses are of prime importance for this life threatening neoplasm. Although immunohistochemical methods provide valuable information such as the location and semi-quantitative data of the interested antigens in particular tumors, conventional methods like histopathological diagnosis remain useful and necessary for identification and classification of tumors. In the present study, we combined histopathology with immunohistochemical staining of estrogen receptors (ER) and progesterone receptors (PR) in canine mammary gland tumors. Fifty dogs with primary mammary tumors underwent surgery at the Veterinary Teaching Hospital of Mahidol University during 2005 to 2007. Three of them were diagnosed with precancerous lesions and negatively stained for ER or PR antibody. Twenty-one were diagnosed with benign tumors classified as adenomas and benign mixed mammary gland tumors. Nearly 60% of thelesions were negatively stained for ER or PR. PR positively stained, both PR and ER stained and ER stained tumors accounted for 19%, 19% and 5%, respectively. Of the malignant tumors, eighty-six percent were adenocarcinomas and 14% were malignant mixed mammary gland tumors. Nearly 70% were negatively stained for ER or PR, 14% were PR positively stained, 14% were both PR and ER stained and 5% were ER stained. Four dogs had unidentified lesions. In summary, more than half of of our benign and malignant canine mammary tumors were negatively stained for ER and PR. This indicates a lack of correlation with estrogen and/or progesterone receptor expression.