A critical assessment of sequencing markers is desirable to ensure that they are appropriate for the specific questions that are to be addressed. This consideration is particularly important where the data set will be used in highly sensitive analyses such as molecular clock studies. However, there is no standard practice for marker assessment. We examined the mitochondrial DNA sequences of a genus of marine molluscs to assess the relative phylogenetic signal of a number of genes using an extension of splits-based spectral analysis. With a data set of almost 8 kb of DNA sequences from the mitochondrial genome of a lineage of marine molluscs, we compared the phylogenetic information content of six protein coding, two ribosomal DNA, and 12 transfer RNA genes. Split-support graphs were used to identify which genes contributed a relatively low signal-to-noise ratio of phylogenetic information. We found that cox2 and atp8 did not perform well for reconstruction at the within-genus level for this lineage. Consideration of nested subsets of taxa improved the resolution of relationships among closely related species by reducing the time frame over which evolutionary processes have occurred, allowing a better fit for models of DNA substitution. Through this fine-tuning of available data it is possible to generate phylogenetic reconstructions of increased robustness, for which there is a greater understanding of the underlying signals in the data. We recommend a suitable mitochondrial DNA fragment and new primers for intergeneric studies of molluscs, and outline a general pipeline for phylogenetic analysis. © 2011 The Linnean Society of London, Biological Journal of the Linnean Society, 2011, 104, 770–785.