Fluorescence resonance energy transfer (FRET)-PCR is widely recognized for its high sensitivity and specificity in pathogen detection. However, there are some gaps in probe design when it is applied for simultaneous detection and differentiation of similar targets. This study aims to investigate the effects of the numbers and position of nucleotide mismatches (NM) in probe on PCR efficiency and melting temperature (T_m). The results indicated that NM at the center reduces amplification efficiency and T_m more significantly than NM at the 5′-terminal or 3′-terminal of the probe.