The karyotype for Puccinia graminis f.sp. tritici was determined from reconstructions of electron micrographs of serially sectioned pachytene nuclei. Epifluorescence microscopy was used to select DAPI-stained, pachytene nuclei in teliospore protoplasts from which walls were mechanically removed. Selection increased the probability that pachytene nuclei could be found in the absence of morphological markers. Six pachytene nuclei were reconstructed from four geographically disparate North American isolates. One nucleus was used to obtain a computer-enhanced, three-dimensional reconstruction that could be rendered as rotatable colorized stereo pairs. A karyotype of n = 18 was determined for all six nuclei. The 18 bivalents varied only slightly in size, each ranging from 3.0 to 8.8% of the total length of the genome. Total genomic lengths also proved highly comparable among isolates. Centromeres were not found, precluding use of the centromeric index as an aid in karyotyping. Only the nucleolus-associated bivalent could be cross-correlated among the six reconstructed nuclei. Heterogeneity in length among, but not within, isolates was observed for this bivalent, suggesting that chromosome length may be polymorphic in P. graminis f.sp. tritici, despite a constant total number of chromosomes. Key words: chromosomes, heterobasidiomycete, synaptonemal complex, Uredinales.