The alkaloids of leaves and flowers of 18 populations of four Pleurothallis (Orchidaceae) species were characterized. Both leaves and flowers of individuals from all populations have two diasteroisomers of 1-hydroxymethylpyrrolizidine (RI=1290 and 1311). The 1290 isomer was the most abundant in all populations of P. johannensis (74-93%) and P. fabiobarrosii (63-93%), two morphologically closed species. On the other hand, in almost all populations of P. teres the 1311 isomer is the more abundant (61-95%), except in one population occurring disjunct from the core area distribution of the species. These results support the recent taxonomic revision for these three species. Almost all P. ochreata populations also have the 1290 as the most abundant isomer, except in one morphologically differentiated population situated at the southern extreme of the distribution of this species.