Brucellosis is a zoonotic disease with veterinary, public health, and economic implications. The study aimed to estimate the seroprevalence of Brucella spp. among ruminants in Rafha, Saudi Arabia during January to October 2022 and to identify camel strains based on a glycosyltransferase gene sequence. Sera (n=1012) were collected from non-vaccinated sheep, goats, camels and cattle of different sex, age and breed randomly from the abattoirs to investigate the circulating brucella antibodies using RBPT. One hundred and eighteen sera (9.7%) were reactive for Brucella spp. IgG immunoglobulins, with higher percentages detected in sheep (11.4%), females (13.3%), adults (10.7%), and naieme breed (13.9%). Significant correlation between Brucella spp. antibodies and animal species (0.095), age (0.077) was found, while strong correlation between antibodies and sex was observed. Glycosyltransferase gene was amplified and sequenced from camel reactive sera (n=6). Camel strains displayed multiple nucleotide substitutions and deletions, nucleotide identity among local strains is 96.2-100%. Phylogenetic analysis showed that Brucella spp. strains clustered in two groups, Rafha strains clustered in one group together with other strains. Further investigation is needed to determine the prevalence of the bacteria among farm animals and to identify the strains involved to improve the preventive measures and strategies adopted for control.