Despite the eradication of brucellosis from most of Europe, the disease remains relatively common in a variety of livestock in southern European countries. It is therefore surprising that with such high prevalence rates, there have been few genetic characterizations of brucellosis outbreaks in this region. We conducted a genetic assessment of 206 isolates of Brucella abortus and B. melitensis from Italy using Variable Number Tandem Repeats (VNTRs). We determined genetic diversity and geographic distribution of these Brucella VNTR genotypes from 160 farms in eight regions of Southern Italy in a fine-scale analysis using 16 VNTR loci in a MLVA-16 methodology. In a broad scale analysis, we then used a reduced dataset of 11 VNTR loci (MLVA-11) to compare genotypes from Italy to a global database. In the 84 isolates of B. melitensis, there were 56 genotypes using MLVA-16; 43 of these genotypes were found only once. At a broad scale, 81 of these isolates were part of an Italian sub-group within the West Mediterranean group. One of the two B. melitensis isolates from a human patient shared the same genotype as a livestock isolate, suggesting a possible epidemiological connection. In 122 B. abortus isolates, there were 34 genotypes by MLVA-16; 16 of these genotypes were found only once. At a broad scale with MLVA-11, one genotype was predominant, comprising 77.8% of the isolates and was distributed throughout Southern Italy. These data on the current lineages of Brucella present in Italy should form the basis for epidemiological studies of Brucella throughout the country, while placing these strains in a global context.