The purposes of this study were to determine the prevalence of heifer clinical mastitis (HCM) and heifer subclinical mastitis (HSCM), isolate HCM-causing bacteria, and assess the association of some risk factors in Huanggang, Central China. A total of 1,374 lactating heifers from three dairy farms were examined in the present study; 22.64% of heifers were positive for mastitis, out of which, 3.86 and 18.78% were with HCM and HSCM, respectively. Of the 67 HCM samples, 91.05% were single growth, 7.46% mixed growth, and 1.49% no bacterial growth. Coagulase-negative staphylococci (CNS) accounted for 30.98% of the isolates followed by Escherichia coli (29.58%), Staphylococcus aureus (16.9%), and Streptococcus uberis (11.27%). Logistic regression analyses showed that heifer milk yield lower than 3 L had the highest prevalence of HCM compared to heifer milk yield of more than 3 L (P < 0.01), and prevalence of HCM was significantly high in heifers with the presence of teat lesions (P < 0.01). Moreover, the heifer milk yield lower than 3 L had the highest prevalence of HSCM compared to heifer milk yield of more than 3 L (P < 0.01). These two factors were significantly associated with the occurrence of heifer mastitis, which needs to be considered in the prevention and control strategies of the disease.