The present study was accentuated upon karyotype analysis and meiotic chromosome behavior in Asteracantha longifolia (L.) Nees. The chromosome number of Astercantha longifolia (L.) Nees-a medicinal herb-found to be 2n = 2x = 32 through mitotic and meiotic analysis. Karyotype analysis revealed eight (6Am + 4Bsm+4Cst+4Dm+4Esm+4Fst+2Gt+4Hsm) morphologically distinct chromosome types. Karyotype analysis showed prevalence of chromosomes with submedian and median primary constrictions. The chromosome length varied from 1.43 to 3.32 μm. The karyotype was symmetric in nature with TF% = 32.8. Meiotic analysis showed the gametic number n = 16 consistently with an average of 15.65±0.65 II and 0.7±0.98 I. The meiocytes revealed predominance of ring bivalents (10.05±0.76) over rod bivalents (5.6±0.68), which had mostly terminalised chiasmata with terminalization coefficient of 0.89. Anaphase-I segregation showed normal 16:16 separation to two poles, although univalents in the form of laggards were often observed. Pollen stainability was 80.3±8.63% indicating the partial heterozygosity in constituent genome of A. longifolia.