Allium cepa L. and Lactuca sativa L. are two model plant species used in studies of environmental pollutants risk assessments. The present study aimed to evaluate the effect of three antimitotic agents on the chromosome complement of A. cepa L. and L. sativa L. and compare the suitability of each one as model for evaluate the effects of environmental pollutants or natural toxic products in the chromosome morphology and cell cycle. To obtain the c-metaphase for the karyotype study, seeds of both species were germinated under controlled temperature, without photoperiod. The obtained roots (up to 2 cm) were treated with three antimitotic agents (colchicine, 8-hydroxyquinoline (8HQ) and cycloheximide) and then fixed in Carnoy. The slides were prepared by squash technique and stained with Schiff's reactive. Metaphases with chromosomes well spread and also individualized presenting defined centromeres were used for karyotype analysis. The A. cepa chromosomes were predominantly metacentric and the size of its chromosomes varied according to the antimitotic applied: 8.74 to 13.44 μm when treated with colchicine, 5.77 to 9.18 μm for 8-HQ and 7.71 to 12.00 μm for cycloheximide. They were categorized as large (L) chromosomes. The chromosomes of L. sativa were predominantly submetacentric and the size of them ranged from 2.60 to 5.30 μm (colchicine), 1.76 to 3.49 μm (8-HQ) and 1.46 to 3.31 μm (cycloheximide), having been classified as small (S). Antimitotic agents caused varying degrees of condensation in the chromosomes of the two species. Under the concentration and exposure time tested, cycloheximide proved to be the antimitotic agent best suited for obtaining best quality C-metaphase in A. cepa, with spread, adequately condensed chromosomes and with well-defined morphology. For L. sativa, the best results were obtained with 8-HQ. Apart from this, A. cepa presents karyotype more homogeneous contributing for a more confident assessment in cytogenetoxic studies.