In order to evaluate the cold tolerance of Lancelot Plantain plant, a Factorial experiment based on completely randomized design was conducted with three replications under controlled conditions at Faculty of Agriculture, Ferdowsi University of Mashhad. Experimental factors included plantain ecotypes (Bojnord, Kalat, Mashhad, Ghayen and Birjand) and freezing temperatures (0, -3, -6, -9, -12, -15, -18 and -21°C). To apply freezing temperatures, Plantain plants were transferred to a Thermo gradient freezer on January 20, 2012, after 3 months growth and hardening under natural environment. In order to assess the plasma membrane stability, electrolyte leakage percentage (EL %) and the lethal temperature required for 50% of plants according to the EL% (LT_50el) was determined. Plant survival assessed four weeks after freezing treatment by measuring survival percentage and the lethal temperature required for 50% of plants according to the survival% (LT_50su). Results showed that in terms of LT_50el, the Mashhad ecotype was the most tolerant and Kalat and Birjand ecotypes were the most sensitive ones. Ecotype and temperature interactions had noticeable effects on EL and Survival percentage, so that Bojnord and Birjand ecotypes had the least and the most EL% at -12 °C, respectively. At the same treatment, Mashhad ecotype with 94.4 % and Birjand ecotype with 25.4% exhibited the most and the least survival percentage. The value of LT_50su forMashhad ecotype was 5.3°C lower than Birjand ecotype. There was high and negative correlations between EL% and survival percentage (r=-0.70^**) in this survey. Moreover, there was significant and positive correlations between LT_50el and LT_50su (r= 0.53^*). According to these findings, Mashhad and Bojnord ecotypes exhibited greater freeze tolerance than Birjand ecotype.