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Background: Taxonomic analysis provides a basic understanding for taxon identification and contributes to preliminary information for several branches of applied biology, while studies on reproductive strategies and plant fitness are essential to interpret population status and dynamics. Aims: We tested the reliability of diagnostic characters for identification and to characterise sexual resource allocation, the breeding system and seed predation among subspecies of Gentiana lutea. Methods: We analysed morphological characters in 70 herbarium specimens. In five natural populations we counted pollen and ovule numbers, assessed reproductive output after pollination treatments and evaluated pre-dispersal predation. Results: Taxonomic traits previously indicated as diagnostic were not sufficient to discriminate among subspecies. The pollen number and pollen:ovule (P:O) ratio varied strongly among subspecies; self-pollinated flowers produced a significantly lower number of seeds than open-pollinated flowers. Retention of empty fruits and high levels of pre-dispersal seed predation were observed in every case. Conclusions: The variation of P:O ratios among subspecies suggests different efficiency in pollen transfer. The species is self-compatible, nevertheless all subspecies require pollen vectors to enhance cross pollination and viable seed production. Fruit retention may have evolved as a strategy to reduce predation, ensuring higher plant fitness.