The viability of six serovars of Leptospira spp. was studied after long storage at −70°C. The bacteria were either preserved in Ellinghausen–McCullough–Johnson–Harris (EMJH) liquid growth medium or in sheep blood added as a cryoprotectant. The viability of the strains was observed on a monthly basis by dark-ground microscopy over a period of 20 months at −70°C. Addition of sheep blood was not significantly advantageous, since leptospires that were stored in EMJH showed a slight increase in number after recovery. The results suggest a very simple and useful technique for long-term preservation of such Leptospira.