BACKGROUND: There are no studies belong NOTCH2 gene polymorphism in relation to reproductive and productive traits in Holstein cattle. The objective of the present study was to investigate the effect of NOTCH2 gene polymorphisms on productive and reproductive performance of fertile and anestrum cattle. METHODS: The cattle were classified into anestrus for 3-12 months postpartum (n = 115, 37.7%) and normal cyclic fertile (n = 190, 62.3%). Age at 1st calving, calving interval, calf birth weight, milk yield and days of milking data were obtained from the farm records for all animals. Blood samples were collected from fertile and anestrum animals for DNA extraction and the PCR as well as single-strand conformation polymorphism techniques (SSCP) were performed for detection of the NOTCH2 gene polymorphism. RESULTS: The sequencing results showed that NOTCH2 gene in cattle is polymorphic with 5 genotypes AA, AG, GG, CC and CT. Four novel SNPs as transition single base substitution mutations were detected at 3: 23,192,033 (A/G), 3:23192149 (C/T), 3:23192071 (C/T) and 3:23192143 (G/A). GG genotype of NOTCH2 gene was associated with shorter calving interval and heavier calf birth weight in both fertile and anestrum cattle. CONCLUSIONS: NOTCH2 gene in cattle could be used as a genetic marker involved in reproductive traits in both fertile and anestrum cattle.