Methods for cluster analysis of IS6110 based restriction fragment length polymorphism (RFLP) patterns of Mycobacterium tuberculosis isolates were studied for an epidemiological investigation in Chiba prefecture. To normalize patterns, external size markers were adopted instead of typical internal size markers used in the standard method. RFLP patterns were run on 1.4% agarose gels and external markers were applied to outside and middle lanes on each gel for precise comparison. The resulting RFLP patterns of 74 isolates were clustered by similarity. Similarity was calculated with the Dice coefficient using parameter settings at 0.8% tolerance and 0.5% optimization. Patterns of 19 isolates from 8 outbreaks showed high similarity within each outbreak. Cluster analysis, as described here, provides insights into epidemiological tracing of tuberculosis in Chiba prefecture.