Streptococcus uberis isolates (n = 55) were obtained from milk samples of cases of mild clinical mastitis in 55 dairy cows from 35 herds serviced by one veterinary practice in Mayenne, France. Isolates were tested for macrolide and lincosamide resistance by using phenotypic and genotypic methods. Erythromycin resistance was detected in 12 of the 55 (22%) isolates based on agar disc diffusion testing and MIC measurements, and was encoded by ermB. This gene also conferred phenotypic resistance to pirlimycin based on MIC measurements, but the D-test was needed for detection of the resistance phenotype in the agar disc diffusion test. Isolates with ermB were also highly resistant to the macrolide antibiotic spiramycin. Seventeen of the 55 isolates (31%) were classified as resistant to spiramcyin only and as having intermediate susceptibility to spiramycin based on agar disc diffusion testing and MIC measurements, respectively. The genetic mechanism behind this phenotype and its clinical relevance are unknown. The efflux pump gene mefA was not detected in any of the 55 isolates in this study. Pirlimycin resistance without macrolide resistance was encoded by the lincosamide resistance gene linB in 4 isolates. Based on current guidelines, some linB-positive isolates would be classified as susceptible by using phenotypic tests, and alternative values for the interpretation of the agar disc diffusion test are suggested. We conclude that the agar disc diffusion test is a useful indicator for macrolide and lincosamide resistance of Strep. uberis in veterinary practice, provided that the D-test is used for detection of pirlimycin resistance.