Animal brucellosis is a reemerging disease in China, particular in northwest China. The Brucella species (even genus) are highly conserved; therefore the use of Multilocus sequencing typing (MLST: based on conserved housekeeping loci) is more suitable for discrimination at species or biovar level on Brucella. In this study, MLST was used to analyze the characterization of Brucella from sheep and yaks during 2015 and 2016. All 66 isolates were collected from northwest China, including Inner Mongolia, Xinjiang, Qinghai, and Gansu provinces. Isolates were cultured on Brucella agar medium and identified by MLST. MLST identified five ST types: ST8 (n = 55), ST7 (n = 2), ST3 (n = 5), ST1 (n = 2), and ST14 (n = 2). This analysis revealed that B. melitensis isolates exhibited high single genotypes (ST8) in the most northwest China. MLST of isolates provides helpful information on understanding genetic characterization of Brucella in northwest China.