The diversity and host specificity of the cyanobionts of several cycad species (Cycas circinalis L., C. rumphii Miq., Encephalartos lebomboensis I. Verd., E. villosus Lem., and Zamia pumila L.) collected in a botanical garden were examined using the tRNALeu(UAA) intron sequence as a genetic marker. Nested PCR was used to specifically amplify the tRNALeu(UAA) intron directly from the freshly isolated symbiotic cyanobionts. By direct amplification of the biological material the laborious isolation of the cyanobionts can be avoided. A single DNA fragment, ranging in size from 287 bp to 329 bp, was consistently amplified from the different biological samples. The intron sequences obtained from the cycad cyanobionts show high similarities to the corresponding sequences in the free‐living strains Nostoc sp. strain PCC 73102 and N. muscorum as well as in several lichen cyanobionts. Although different Nostoc strains were found in the present study, no sequence variation was observed when analyzing a single coralloid root. However, different coralloid roots from a single cycad individual may harbor different cyanobacteria. Moreover, cyanobionts in coralloid roots of both Encephalartos lebomboensis and E. villosus were found to possess the same intron sequence.