Despite the recent discovery of interchromosomal telomere length variation, a role for heterogeneity in telomere maintenance has yet to be established. This study aimed to clarify relative telomere length differences between chromosomes. Combined chromosomal sorting and telomeric repeat content analysis in GM130B cells, the relative telomeric repeat content in each chromosome, were calculated. Each chromosome could be isolated except for chromosomes 1 and 2 and chromosomes 9 to 12, which were isolated in a group. Telomere length was correlated with the size of the corresponding chromosome. Concomitant relative telomeric repeat content analysis in each chromosome and terminal restriction fragment analysis using the whole genome revealed that the terminal restriction fragments of each chromosome were heterogenously distributed through the smear of the fragments. This is the first description of an association between telomere length and chromosome size.