Commercial dairy farming in Pakistan is rapidly growing. Crossbred dairy animals hold promise because of two reasons, first that they are significantly high producer than buffalo, Sahiwal, Cholistani and Desi cows and secondly that they are much more adaptable as compared to exotic (Friesian or Jerseys) cows. Generally, replacement heifers remain ignored by the owners in term of feeding and early achievement of puberty as they are not producing. Estradiol promotes tighter synchrony and ovulation and provides opportunity for fixed time A.I as reported earlier in Holstein dairy cows. Use of CIDR has been reported to be effective for synchronization in variety of dairy animals including buffaloes, dairy and beef cows. However no such report has been published on crossbred heifers. The objectives of the present study were to investigate the effect of estradiol benzoate (EB) on estrus intensity, response and fertility in CIDR treated cross bred heifers.Crossbred heifers with average body condition of 3 ± 0.5, located at four farms were assigned throughout one year to receive one of the two treatments. Crossbred heifers with EB (n = 50) or without EB (n = 50). All the heifers were treated with Controlled Internal Drug Releasing Device (CIDR; 1.38 mg Progesterone; Pfizer, USA) on day 0 and were administered injection of PGF2?, (0.150 mg; Dalmazine®, Fatro, Italy; 2 ml; i.m.) on day 6 followed by removal of CIDR on day 7. Estradiol benzoate (400µg/4ml, i.m. Sigma Aldrich Germany) was administered 24 h after the CIDR removal. Estrus detection was carried out by visual observation; twice daily for at least 30 min, beginning after CIDR removal till insemination. All heifers were fixed time inseminated with frozen-thawed semen after 48 and 60 h after CIDR removal, respectively. Pregnancy status was determined by ultrasonography (HS 1500V; Honda; Japan, with 7.5 MHz probe) between days 30 and 40 post AI. Estrus response and pregnancy rate were analyzed using Chi-square test. Pregnancy rate was determined by number pregnant/number treated x 100. The estrus response was 100% in both the groups. The estrus intensity was higher 2.9 ± 0.1in the EB heifers compared to the controls 2.0 ± 0.7. The results showed that pregnancy rate was 54% (27/50) in EB and 36% (18/50) in without EB heifers. The overall pregnancy rate was 45% (45/100). It is concluded that estradiol benzoate in CIDR protocol intensified the signs of estrus without affecting the response, with higher fertility in crossbred heifers.