For the aim of genotypic fingerprinting of Brucella melitensis bv3 isolated from different ruminant species in Kafrelsheikh governorate, Egypt, a multilocus variable-number tandem-repeat analysis (MLVA 16) has been approached. The MLVA 16 was performed on 41 B. melitensis bv3 isolates identified by bacteriological and molecular techniques. Thirty-one isolates originated from the preferential host (28 sheep and three goats), and ten isolates from atypical hosts (nine cattle and one buffalo). Recovering the same genotype in two different animal species suggests cross-species adaptation of B. melitensis bv3 to different atypical ruminant species in Egypt. Furthermore, the isolation of B. melitensis from aborted cows after the entry of a replacement cow from an unknown brucellosis status herd in cattle farms that had never reared small ruminants indicates that cows can be infected and spread the infection without the presence of the original host. Our results further showed that different genotypes of B. melitensis could be isolated from different samples of the same animal. The local geographic distribution of genotypes showed a very close genetic relatedness with previously reported genotypes outside the study area. Worldwide, all genotypes and strains identified in this study were mostly related to the Western Mediterranean lineage and were less likely to the Americas clonal lineage. In conclusion, uncontrolled animal movement and the ability of B. melitensis to spread among atypical hosts in the absence of the original hosts are potential causes for the failure of brucellosis control programs in endemic areas. The legal importation and illegal movement of cattle and sheep are the main factors for maintaining the infection of B. melitensis within the country. Further investigations are required to understand the reasons for the presence of more than one genotype of B. melitensis in the same animal and the efficacy of the current applied strategy for brucellosis control.