Microspore culture of wheat generates completely homozygous (doubled haploid) plants in a single generation thereby reducing the time required for wheat variety development. Success of microspore culture in spring wheat is relatively higher than that in winter wheat. Cold mediated pretreatment was reported to improve response of microspore culture in wheat. The objective of the study was to determine and compare the influence of cold pretreatment on microspore culture in spring and winter wheat. Three spring (“Chris”, “Express”, and “Macon”) and three winter (“Anton”, “Antelope”, and “Camelot”) wheat cultivars were used. In cold pretreatment, excised anthers were incubated in solution B at 25°C-28°C for 4-5 days followed by cold treatment at 4°C for 5 days and were compared with the no-cold pretreatment at 25°C-28°C for 4-5 days. Isolated microspores were cultured in induction medium (MMS4) at 27°C-28°C for 25-30 days in the dark. Embryos (1-2 mm size) were transferred to regeneration medium (MMS5). Numbers of multicellular structures, transferable embryos and green plants were counted and data were used for analysis of variance using a generalized linear model. It was observed that cold pretreatment increased multicellular structures, transferable embryos and green plants in both spring and winter wheat. However, the degree of improvement was higher in spring wheat compared to winter wheat. The cultivars within spring and winter wheat responded differently. Development of embryos from pro-embryos was 4-5 folds lower in winter wheat than that in spring wheat, indicating requirement of a possibly different hormonal composition in induction medium for improving embryo induction in winter wheat. This report may provide future direction of research to improve microspore culture response in winter wheat.