Rapid production of doubled haploids (DHs) through isolated microspore culture is an important and promising method for genetic study of alfalfa. To induce embryogenesis in alfalfa, isolated microspores were submitted to abiotic stresses during their initial culture, in order to stimulate them to form embryos and plantlets. 'Baoding' and 'Zhongmu No 1' alfalfa cultivars supported reproducible and reliable proliferation response irrespective of any stress treatment of microspores. The microspore developmental stage for isolated microspore culture was studied and we found that uninucleate microspores were best to initiate culture. Exposure of microspores to appropriate low temperature or heat shock stresses were able to increase the efficiency of embryogenesis. The most effective low-temperature treatment was 4 °C for 24 h and the frequency of plantlets induction was 20.0%. The most effective heat shock treatment was 32 °C for 2 d and the frequency of plantlets induction was 14.17%. The analysis of ploidy level performed by flow cytometer revealed that the majority of 278 regenerated plantlets were haploid (65.83%) or doubled haploid (33.81%). This is the first report of haploid production in alfalfa through isolated microspore culture.