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<i>Streptococcus uberis</i>, an environmental pathogen responsible also for contagious transmission, has been increasingly implicated in clinical mastitis (CM) cases in Europe. We described a 4-month epidemiological investigation of <i>Strep. uberis</i> CM cases in an Italian dairy farm. We determined molecular characteristics and phenotypic antimicrobial resistance of 71 <i>Strep. uberis</i> isolates from dairy cows with CM. Genotypic variability was investigated via multiplex PCR of housekeeping and virulence genes, and by RAPD-PCR typing. Antimicrobial susceptibility was assessed for 14 antimicrobials by MIC assay. All the isolates carried the 11 genes investigated. At 90% similarity, two distinct clusters, grouping 69 of the 71 isolates, were detected in the dendrogram derived from the primer ERIC1. The predominant cluster I could be separated into two subclusters, containing 38 and 14 isolates, respectively. <i>Strep. uberis</i> strains belonging to the same RAPD pattern differed in their resistance profiles. Most (97.2%) of them were resistant to at least one of the drugs tested, but only 25.4% showed a multidrug resistance phenotype. The highest resistance rate was observed for lincomycin (93%), followed by tetracycline (85.9%). This study confirmed a low prevalence of β-lactam resistance in <i>Strep. uberis,</i> with only one isolate showing resistance to six antimicrobial classes, including cephalosporins.