Alfalfa (Medicago sativa L.) is the one of the important and economic forage legume crops in the world and Turkey. Alfalfa has 60 species which 30 annual and perennial species were distributed in different regions of Turkey. Cytogenetical research on alfalfa lagged far behind other crops mainly because alfalfa chromosomes are very small, the chromosomes are morphologically very similar, cultivated alfalfa has relatively high number of chromosomes and alfalfa is an autotetraploid. The Yesilova-3 alfalfa population was collected from Lake Regions of Turkey as clone. Root tips meristems obtained from plants of similar age that originated by vegetative reproduction from a single parent from plants grown under greenhouse conditions, were pretreated with saturated solution of alpha-bromonaphthalene and 0.002 M 8-Hydroxiquinolin before staining with aceto-iron-hematoxylin. The measured chromosomal parameters were; Short arm, Long arm, Total length of chromosomes, Arm Ratio and chromosome type. Within the chromotosome complement, seven pairs of chromosomes were metacentric (chromosomes II, III, V and VI) and three numbers were submetacentric (chromosome I, IV and VIII). Karyotype analysis showed that Yesilova accession was tetraploid (2n=4x=32). The short arm, long arm and total length of chromosomes were ranged from 0.82-1.22, 1.17-1.80 and 2.21-2.85 micrometer, respectively.