Abstract Background Heifers can calve down with intramammary infections (IMI) and udder damage. This will have a negative impact on their longevity, future milk yield and financial return. Co-housed pre-weaned calves that are fed fresh milk have the opportunity to suckle each other’s teats and may infect udders of fellow heifer calves with pathogens present in milk. The prevalence of IMI in pregnant heifers in South Africa (SA) which were co-housed and reared on fresh milk as calves, is not known. Quarter secretion samples from both pregnant heifers (n = 2065) and dry cows (n = 5365) were collected for microbiological analysis from eight SA dairy herds. All heifers tested in this study were co-housed pre-weaning and were fed fresh milk as calves. Results The prevalence of coagulase negative staphylococci, Staphylococcus aureus, Streptococcus agalactiae, environmental streptococci, coliforms and samples with no bacterial growth in heifers was 26%, 0.9%, 0.08%, 1.4%, 0.4% and 66%, respectively. The overall prevalence ratio between heifers and cows for Staphylococcus aureus IMI was 0.76 (95% CI: 0.59, 0.98). Four of the individual herds had prevalence ratios of less than one (p < 0.05), one herd had a prevalence ratio of 3.15 (95% CI: 1.52, 6.32), and the remaining 3 herds had a prevalence ratio not significantly different from 1.0. Marginally significant differences were found between Staphylococcus aureus IMI in pregnant heifers compared to cows in their second and later lactations (p = 0.06, p = 0.05, respectively) but no significant differences between heifers and cows in their first lactation. Conclusions The presence of Streptococcus agalactiae IMI in heifers came as a surprise, especially as herd infection rates were low. The high prevalence ratio of Staphylococcus aureus between heifers and cows in one herd warrants further investigation due to the potential danger of udder damage in a young cow at the start of her productive life. The IMI in heifers with host adapted pathogens can also act as a source of new IMI for lactating dairy cows.