Repetitive sequence-based PCR (rep-PCR) is a potential epidemiological technique that can provide high-throughput genotype fingerprints of heterogeneous <i>Mycobacterium</i> strains rapidly. Previously published rep-PCR primers, which are based on nucleotide sequences of Gram-negative bacteria may have low specificity for mycobacteria. Moreover, it was difficult to ensure the continuity of the study after the commercial rep-PCR kit was discontinued. Here, we designed a novel rep-PCR for <i>Mycobacterium intracellulare</i>, a major cause of nontuberculous mycobacterial pulmonary disease with frequent recurrence. We screened the 7,645 repeat sequences for 200 fragments from the genome of <i>M. intracellulare</i> ATCC 13950 <i>in silico</i>, finally generating five primers with more than 90% identity for a total of 226 loci in the genome. The five primers could make different band patterns depending on the genome of three different <i>M. intracellulare</i> strains using an <i>in silico</i> test. The novel rep-PCR with the five primers was conducted using 34 bacterial samples of 7 species containing 25 <i>M. intracellulare</i> clinical isolates, compared with previous published rep-PCRs. This shows distinguished patterns depending on species and blotting assay for 6 species implied the sequence specificity of the five primers. The Designed rep-PCR had a 95-98% of similarity value in the reproducibility test and showed 7 groups of fingerprints in <i>M. intracellulare</i> strains. Designed rep-PCR had a correlation value of 0.814 with VNTR, reference epidemiological method. This study provides a promising genotype fingerprinting method for tracing the recurrence of heterogeneous <i>M. intracellulare</i>.