The aim of the present study is to determine variations of estrone sulphate (SO4E1), progesterone (P4), 17-beta estradiol (E2), testosterone (T) and prolactin (PRL) levels in serum, mammary tissue homogenates as well as variations in the distribution of androgen (AR), estrogen alpha (ER alpha), progesterone (PR) and prolactin (PRL-R) receptors in mammary tumours of dogs. Thirty bitches from different breeds and ages with mammary lesions (experiment group) and 10 healthy bitches (free of any mammary lesions=control group) were used in this study. All the dogs included to the study were in anoestrus. Mammary lesions of experiment group were surgically removed and 10 normal mammary tissue samples were collected by surgical biopsy from control group dogs. Fifty-seven mammary tissue samples were obtained and were divided into two fragments for histopathology/immunohistochemistry and for hormone determinations in tissue homogenate. Besides, blood samples were collected from all dogs before the surgeries to detect hormonal variations. Student t-test was used for serum samples, one way ANOVA and tukey test were used in tissue homogenate for statistical analysis. According to current study results; PRL levels in serum were found significantly higher (P<0.01) in the experiment group compared to the control group. In tissue homogenate samples, T levels were found to be significantly (P<0.01) higher in malignant mammary tissue samples. PR was intensely expressed in 23, PRL-R was expressed in 13, ERa was expressed in 9 and AR was expressed in 6 cases of canine malignant mammary tumours (CMTs). In conclusion, PRL levels in serum, T in tissue can act on the formation of canine mammary tumours which may have different features of developments in different mammary glands of the same dog and with the predominance of PR expression in CMT.