An 11-month-old Holstein heifer diagnosed as a female pseudohermaphrodite (PH) was subjected to clinical, hormonal, histological, and cytogenetic examinations. The urogenital sinus and external genitalia of the heifer were virilized and no vulval orifice was present. A male urethra-like tubular structure was palpable in the perineal area; urination occurred through a urethral fossa at the distal end of the urethra (cranial aspect of the mammary tissue). Bilateral ovaries and uterine horns, ovarian follicular development, ovulation and CL formation (with increasing blood P4 concentration) were similar to a normal heifer. Testosterone (T) concentrations of all samples collected both before and after hCG challenge were <0.1 ng/mL (absence of a responsive endogenous androgen source). Histologically, the ovary, uterus, oviduct and vagina resembled the normal female reproductive tract. Although the corpus cavernosum was present, the penile urethra and corpus spongiosum penis were hypoplastic. A normal female karyotype (60, XX) was detected in metaphase spreads obtained from cultured peripheral lymphocytes. Moreover, Y-chromosome-specific sequences were not detected in genomic DNA from lymphocytes. By PCR analysis of genomic DNA (from the tissues of urogenital organs), Y-chromosome-specific sequences (both SRY and Amelogenin (Amel) locus) were detected. In conclusion, this was a female PH heifer due to gonadal mosaicism, with normal female genitalia and ovarian activity. This is the first report of an SRY-positive, mosaic female PH heifer.