Methicillin-resistant Staphylococcus aureus (MRSA) and methicillin-resistant Staphylococcus pseudintermedius (MRSP) are opportunistic pathogens that cause significant diseases in both human and veterinary medicine, particularly in pet animals. In recent decades, the emergence of MRSA and MRSP in pet animals has raised substantial public health concerns due to the high multidrug resistance exhibited by these zoonotic bacteria. Therefore, a rapid and accurate diagnosis is crucial as one of the key strategies for controlling infections caused by MRSA and MRSP. In this study, we applied a simple and cost-effective DNA extraction method, establishing a multiplex PCR assay for the identification and differentiation of MRSA and MRSP isolated from dogs in Vietnam. The results of this study indicate that DNA extraction of MRSA and MRSP using the Tris-EDTA-NaCl-Triton X100 (TENT) method yielded comparable efficacy to using a commercial DNA extraction kit, and the TENT method proved to be more cost-effective. Additionally, a multiplex PCR assay with four primer pairs (27F/1492R, mecA_F/mecA_R, au-F3/au-nucR, and pse-F2/pse-R5) successfully identified and discriminated between MRSA and MRSP. The multiplex PCR demonstrated high specificity with the desired amplification of targeted amplicons and no cross-reactions. Furthermore, the applicability of the diagnostic procedure was demonstrated by identifying field MRSA and MRSP isolated from clinical samples of dogs in Vietnam. Our study, therefore, provides an effective identification procedure for MRSA and MRSP, serving as a prompt countermeasure against the rapid emergence of MRSA and MRSP in Vietnam