Telomeres are the specialized structures at the ends of eukaryotic chromosomes and are composed of short T/G-rich DNA repeats and the proteins that interact with them. Internal to telomeres are subtelomeric regions that are species-specific and often repetitive. The yeast Kluyveromyces lactis has telomeric tracts of 10–20 copies of a 25 bp repeat, but the subtelomeric regions have not previously been characterized in detail. Here we have cloned and characterized subtelomeric regions from 10 of the 12 chromosome ends. The amount of sequence examined was 0.7–10 kb for each subtelomeric region. We have identified a K. lactis subtelomeric element, the R element, which has a strong purine/pyrimidine strand bias and extends for about 2 kb. Internal to the R element, we found extensive similarity that is shared among half of the chromosome ends reported here. This similarity appears to include three putative gene families, two of which are also subtelomeric in Saccharomyces cerevisiae. GenBank Accession Nos: P1, AY437818; P2, AY437819; P3, AY437820; P4, AY437821; P5, AY437822; P6, AY437823; P7-5′, AY437824; P7-3′, AY437825; P9-5′, AY437826; P9-3′, AY437827; P10, AY437828; P11-5′, AY437829; P11-3′, AY437830. Copyright © 2004 John Wiley & Sons, Ltd.