Glucosinolates are unique thioglucosides that evolved in the order Brassicales. These compounds function in plant adaptation to the environment, including combating plant pathogens, herbivore deterrence and abiotic stress tolerance. In line with their defensive functions glucosinolates usually accumulate constitutively in relatively high amounts in all tissues of Brassicaceae plants. Here we performed glucosinolate analysis in different organs of selected species representing Capsella, Camelina and Neslia genera, which similarly as the model plant Arabidopsis thaliana belong to the Camelineae tribe. We also identified orthologs of A. thaliana glucosinolate biosynthetic genes in the published genomes of some of the investigated species. Subsequent gene expression and phylogenetic analyses enabled us an insight into the evolutionary changes in the transcription of these genes and in the sequences of respective proteins that occurred within the Camelineae tribe. Our results indicated that glucosinolates are highly abundant in siliques and roots of the investigated species but hardly, if at all, produced in leaves. In addition to this unusual tissular distribution we revealed reduced structural diversity of methionine-derived aliphatic glucosinolates (AGs) with elevated accumulation of rare long chain AGs. This preference seems to correlate with evolutionary changes in genes encoding methylthioalkylmalate synthases that are responsible for the elongation of AG side chains. Finally, our results indicate that the biosynthetic pathway for tryptophan-derived indolic glucosinolates likely lost its main functions in immunity and resistance towards sucking insects and is on its evolutionary route to be shut off in the investigated species.