Sexual dysfunction following breast cancer treatment is common and screening for this is recommended. This study determined the reliability, validity, and acceptability of a breast cancer-specific adaptation of the Female Sexual Function Index, the FSFI-BC. This new measure addresses limitations in the FSFI when assessing sexual dysfunction of women with breast cancer regarding applicability to non-sexually active women, measuring distress and changes after cancer. Female breast cancer survivors (n = 596; 429 sexually active, 166 non-sexually active) completed an online survey including demographic/medical information, the FSFI-BC, and scales measuring sexual functioning, fatigue, body image, physical and mental health, and relationship adjustment (Time 1). Three weeks later, 326 women (245 sexually active; 81 non-sexually active) completed the Time 2 survey including the FSFI-BC, and questions regarding its acceptability and perceived change in sexual functioning. Reliability, construct validity, and acceptability were examined using standard scale validation techniques. Exploratory factor analysis delineated seven factors: Changes after cancer, desire/arousal, lubrication, orgasm, pain, satisfaction, and distress, accounting for 79.98 % (sexually active) and 77.19 % (non-active) variance in responses. Acceptable internal consistencies (non-active: α = 0.71–0.96; sexually active: α = 0.89–0.96) and test–retest reliabilities (non-active: r = 0.63–0.86; sexually active: r = 0.71–0.88) were evident. Inter-scale correlations provided evidence for convergent and divergent validities of the FSFI-BC. Both sexually active and non-active women provided positive feedback about the FSFI-BC. The optional partner questions demonstrated clinical utility. With desirable psychometric properties and acceptability to participants, the FSFI-BC is suitable for screening for sexual dysfunction in women with breast cancer.