Characterization of a low-copy number DTF1 satellite DNA detected in the bivalve mollusk Donax trunculus revealed extensive grouping of monomer sequence variants into subfamilies identified by distinctive combinations of diagnostic nucleotides. It can be anticipated that a large number of subfamilies exists in the genome. In addition to the tandem organization of 169 bp long monomers, at least one subfamily was created through amplification of adjacent repeats in a higher order register. This complex satellite unit consists of two distinctive monomer variants that differ both in specific nucleotide changes and in a deleted segment partially substituted with a short unrelated sequence element. Most of the nucleotide substitutions differing between subfamilies are highly homogenized within a corresponding group of monomer variants, and intra-subfamily variability in general is low. Nucleotide diversity analysis of all sequenced variants of DTF1 satellite revealed the presence of two conserved segments, while the rest of the monomer sequence shows uniform and considerably higher level of variability. The persistence of conserved segments stands in contrast to the sequence and organizational divergence of monomer variant groups, and may indicate constraints in the evolution of DTF1 satellite repeats.