Leaf and branch samples of 10 Populus nigra L. cv. Italica trees were collected from the urban Zagreb area in late summer/autumn 2001. One of the trees exhibited leaf yellowing, overall sparse foliage, stunting and decline. Phytoplasma 16S rDNA was amplified in direct and nested PCR assays using universal and specific phytoplasma primer pairs, from nucleic acids extracted by two different procedures. Strong amplification signals were observed in samples from symptomatic Lombardy poplar as well as in samples from 4 of the asymptomatic trees. RFLP analyses of amplicons showed patterns characteristic of the phytoplasmas belonging to the Aster yellows group (16SrI). This is the first report of a phytoplasma naturally infecting poplar in Croatia.