Recent advances in molecular technologies have opened up unprecedented opportunities for molecular ecologists to better understand the molecular basis of traits of ecological and evolutionary importance in almost any organism. Nevertheless, reliable and systematic inference of functionally relevant information from these masses of data remains challenging. The aim of this review is to highlight how the Gene Ontology (GO) database can be of use in resolving this challenge. The GO provides a largely species-neutral source of information on the molecular function, biological role and cellular location of tens of thousands of gene products. As it is designed to be species-neutral, the GO is well suited for cross-species use, meaning that, functional annotation derived from model organisms can be transferred to inferred orthologues in newly sequenced species. In other words, the GO can provide gene annotation information for species with nonannotated genomes. In this review, we describe the GO database, how functional information is linked with genes/gene products in model organisms, and how molecular ecologists can utilize this information to annotate their own data. Then, we outline various applications of GO for enhancing the understanding of molecular basis of traits in ecologically relevant species. We also highlight potential pitfalls, provide step-by-step recommendations for conducting a sound study in nonmodel organisms, suggest avenues for future research and outline a strategy for maximizing the benefits of a more ecological and evolutionary genomics-oriented ontology by ensuring its compatibility with the GO.