The objective of this study was to evaluate the expression of herbicide-tolerance in tobacco regenerants from important Bulgarian tobacco cultivars and their seed progeny by cytological and morphological analyses. The herbicide used in this investigation was glyphosate _ nonselective broad spectrum herbicide. Most of the morphological anomalies were accompanied by slow growth, reduced vigour, malformed leaves, flowers and internodes. It was observed that the chromosome number variation was greater at higher glyphosate concentration in the medium. Variants regenerated from the same callus clones showed various chromosome numbers, indicating that intercellular chromosomal heterogeneity existed with clones. The older the callus culture, the greater the degree of ploidy instability observed. Ploidy instability was measured as plants regenerated being either polyploid or aneuploid. Our investigation showed that the regeneration of plants from young glyphosate tolerant callus lines allowed recovery more of 50% putative tolerant plant with normal chromosomal complement, regeneration competence and fertility.