Objective To develop a PCR for the detection of corynebacterium kutscheri （C. kutscheri） and apply it to clinical samples. Methods Genomic DNA was extracted as template for PCR from C. kutscheri (ATCC 11306) recoveried and cultivated in brain heart infusion medium. According to the C. kutscheri 16S rRNA gene sequence available in GenBank a pair of primes were designed and synthesized in order to develop a PCR for detection of C. kutscheri. After evaluated for sensitivity and specificity the PCR method was applied to detect the C. kutscheri in clinical livers and kidneys of mice artificially infected with C. kutscheri. Results The PCR for the detection of C. kutscheri was developed successfully and were specific enough to distinguish C. kutscheri from salmonella, streptococcus pneumoniae and Pasteurella. A minimum of 100 positive plasmids could be detected, indicating a good sensitivity of the assay. Conclusion This method repoted here is specific, sensitive and provides a fast detection of C. kutscheri and could be used for C. kutscheri clinical diagnosis.