We describe the occurrence of a tandem repeat in the chloroplast genome of the marsh orchid, Orchis palustris. The repeat unit is an AT-rich, 16-bp sequence located in the chloroplast tRNALEU intron. Southern blot analysis confirmed that the O. palustris tRNALEU intron including the minisatellite locus has not been transferred to the nucleus, but is indeed located on the chloroplast genome. The 16-bp repeat unit was found to be present in all O. palustris accessions studied, as well as in the closely related O. laxiflora. Variation in repeat numbers among individuals was found in O. palustris from central and northern Italy; and this was consistently associated with a 13-bp sequence motif preceding the repeat. This motif was absent from O. palustris from southern Italy, Greece, and from O. laxiflora. In these accessions, no variation in repeat numbers was found. Our results suggest that the O. palustris chloroplast minisatellite locus evolved relatively recently, presumably in central Italy, and may represent a valuable marker for population genetic studies.