Mitochondrial targeting signals (MTSs) are responsible for trafficking nuclear encoded proteins to their final destination within mitochondria. These sequences are diverse, sharing little amino acid homology and vary significantly in length, and although the formation of a positively-charged amphiphilic alpha helix within the MTS is considered to be necessary and sufficient to mediate import, such a feature does not explain their diversity, nor how such diversity influences target sequence function, nor how such dissimilar signals interact with a single, evolutionarily conserved import mechanism. An in silico analysis of 296 N-terminal, matrix destined MTSs from Homo sapiens, Mus musculus, Saccharomyces cerevisiae, Arabidopsis thaliana, and Oryza sativa was undertaken to investigate relationships between MTSs, and/or, relationships between an individual targeting signal sequence and the protein that it imports. We present evidence that suggests MTS diversity is influenced in part by physiochemical and N-terminal characteristics of their mature sequences, and that some of these correlated characteristics are evolutionarily maintained across a number of taxa. Importantly, some of these associations begin to explain the variation in MTS length and composition.