Comparative analysis of the telomeres of distantly related species has proven to be helpful for identifying novel components involved in telomere maintenance. We therefore initiated such a study in the nonconventional yeast Yarrowia lipolytica. Its genome encodes only a small fraction of the proteins that are typically associated with telomeres in other yeast models, indicating that its telomeres may employ noncanonical means for their stabilization and maintenance. In this report, we have measured the size of the telomeric fragments in wild-type strains, and characterized the catalytic subunit of telomerase (YlEst2p). In silico analysis of the YlEst2 amino acid sequence revealed the presence of domains typical for telomerase reverse transcriptases. Disruption of YlEST2 is not lethal, but results in retarded growth accompanied by a rapid loss of the telomeric sequences. This phenotype is associated with structural changes at the chromosomal ends in the ΔYlest2 mutants, likely the circularization of all six chromosomes. An apparent absence of several typical telomere-associated factors, as well as the presence of an efficient means of telomerase-independent telomere maintenance, qualify Y. lipolytica as an attractive model for the study of telomere maintenance mechanisms and a promising source of novel players in telomere dynamics.