We tested whether proteome reference maps established for one species can be used for cross‐species protein identification by comparing two‐dimensional protein gel patterns and protein identification data of two closely related bacterial strains and four plant species. First, proteome profiles of two strains of the fully sequenced bacterium Sinorhizobium meliloti were compared as an example of close relatedness, high reproducibility and sequence availability. Secondly, the proteome profiles of three legumes (Medicago truncatula, Melilotus alba and Trifolium subterraneum), and the nonlegume rice (Oryza sativa) were analysed to test cross‐species similarities. In general, we found stronger similarities in gel patterns of the arrayed proteins between the two bacterial strains and between the plant species than could be expected from the sequence similarities. However, protein identity could not be concluded from their gel position, not even when comparing strains of the same species. Surprisingly, in the bacterial strains peptide mass fingerprinting was more reliable for species‐specific protein identification than N‐terminal sequencing. While peptide masses were found to be unreliable for cross‐species protein identification, we present useful criteria to determine confident matching against species‐specific expressed sequence tag databases. In conclusion, we present evidence that cautions the use of proteome reference maps and peptide mass fingerprinting for cross‐species protein identification.