Abstract: Heifer mastitis causes detrimental mammary gland development affecting the subsequent lactating stages, udder health and related culling hazard resulting in significant economic losses to the dairy development sector. Across sectional study was conducted from June 2011 to March 2012 on cross breed heifers in Debre-zeit and Sebeta towns to estimate the prevalence of heifer mastitis, isolate bacteria causing mastitis and test their antimicrobial susceptibility. One hundred fifty eight (158) heifers were able to include from one hundred forty nine (149) cooperative smallholder dairy farms during the study period. From the total of 158 heifers sampled, 46(29.1%) were positive for mastitis (9.5% clinical and 19.6% subclinical cases). Identification of the bacteria on primary culture was made on the basis of colony morphology, hemolytic characteristics, gram stain reaction including shape and arrangements of the bacteria, catalase and oxidation and fermentation (o-f) test and further differentiation within the species level were made by selective media. The most frequently isolated bacteria from quarter milk samples in for clinical and subclinical mastitis were 7(24.1%) and 22 (75.9%) CNS, 7(26.91%) and 19(73.1% ) Staphylococcus aureus and 4(22.2%) and 14(77.8%) E. coli, respectively. Other bacterial isolates were Streptococcus agalactiae (1(11.1%) and 8(88.9%), Kelebsella pneumonia (3(37.5%) and 5(62.5%)), Bacillus cerus (1(16.7%) and 5(8.3.3%)), actinomycet pyogens (1(25%) and 3(75%)), Streptococcus dysagalactiae 0 and 3(100%), Entroccoccus feacalise (0 and 3(100%)) and Streptococcus uberis (0 and 3(100%)) for clinical and subclinical mastitis, respectively. The univariate logistic regression showed that among the risk factors considered, age, heifer status, mastitic milk fed to calves, body condition scoring, usage of waste disposal and udder hygiene had significant effect on the prevalence of subclinical mastitis. However, after multivariate analysis, only age (OR = 2.1; CI, 1.5-2.9), mastitic milk fed to calves (OR = 2.3; CI, 1.5-3.4), udder hygiene (OR = 1.9; CI, 1.4-2.5) and usage of waste disposal (OR = 2.7; CI, 1.6-4.4), had significant effect. The antimicrobial susceptibility test showed for the majority of bacterial isolates including the major pathogens had 75-100% susceptibility pattern. CNS and Streptococcus dysagalactiae were the species which showed 100% susceptibility for all of the antimicrobials tested while the remaining species had varying levels of susceptibility (50-100%). Among isolates Staphylococcus aureus show relatively lower susceptibility for almost all antimicrobials used. Streptomycin and Erythromycin was the most effective antibiotic followed by Sulfisoxazole and Ampicillin. The presence of mastitis in heifer in early age indicates important economic losses. Therefore, awareness creation at the smallholder dairy farm on the economic significance of heifer mastitis, risk factors that plays vital role in establishment and flourishment of potential pathogen and use of dry cow therapy before calving will help in reducing mastitis in heifer. Moreover, further studies on what extent the causative pathogen and the host itself affect the persistence of intramammary infection during calving and early lactating heifers and evaluation of other risk factors in depth will merits the dairy farms.