Objective: To investigate the non-specific and inaccurate amplification in cases of highly similar sequences among family members and the length heterogeneity of mature microRNA ( miRNA) ,and find a condition that discriminates maximally among similar miRNA family members and detects the accurate expression level of miRNAs. Methods: Primers with their mismatches and/or 3' end at different positions were designed. Amplification efficiencies were compared using matched and various mismatched primers by RNA-tailing and primer-extension RT-PCR at different annealing temperatures. Expression levels of several miRNAs in mouse brain were compared using miRNA specific primers with different termini. Results: Raising annealing temperatures 12℃-14℃above the T_m of the primers maximally increased amplification specificity without sacrificing sensitivity. Primers designed with their termini on or near variant positions could efficiently discriminate between miRNA isoforms. Using primers that terminated before the end of the mature miRNA did not miss the detection of shorter mature miRNA and provided accurate expression levels. Conclusion: Careful primer design and higher annealing temperature can increase specificity and accuracy of real time PCR miRNA detection.