Bovine viral diarrhea virus (BVDV) is a highly infectious pathogen that affects bovines worldwide leading to great economic impact. Although Brazil has the largest commercial cattle population throughout the world and an increasing buffalo breeding industry, the country has no control or eradication program for BVDV. In this perspective, the aim of this study was to evaluate the occurrence of BVDV in cattle and buffaloes from two Brazilian states. Four different ELISA tests were performed and confirmed by virus neutralization testing (VNT). The presence of BVDV antibodies in the serum or plasma from 77 cattle from six herds (ELISA-1 and ELISA-4) and from 89 buffaloes from three herds (ELISA-1 through ELISA-4) was detected. Extraction of viral RNA was performed from the serum or plasma samples for the detection of BVDV by RT-PCR analysis. Amplified nucleotide sequences were used to construct a phylogenetic tree. In cattle, ELISA-1 detected 49.4% of seropositive animals, while ELISA-4 detected 37.7%. In buffaloes, ELISA-1 failed to detect any seropositive animals, while ELISA-2 and ELISA-3 detected 20.2% of seropositive animals, and ELISA-4 detected 21.3%. Eight of the nine herds tested had seropositive animals. The rate of PCR positive animals was 6.5% in cattle and 9% in buffaloes. Subtype 1d was found in cattle, and subtypes 1d and 1f were found in buffaloes. This is the first- time subtype 1f has been reported in Brazil. The absence of a control and eradication program seems to be favoring the spread of BVDV in the Brazilian herds. In addition, the improvement of diagnostic strategies for BVDV in buffaloes are required.