Reproductive strategies of hibernating insectivorous bats and reproduction phenology parameters can vary considerably as they will be influenced by ecological seasonality. While the exact timing of greater mouse-eared bat (Myotis myotis) reproduction is described through morphological assessment of dissected embryos, most recent studies have focused only on population or behavioural aspects of reproduction. In our study, non-invasive ultrasound examination and non-lethal blood sampling were used to assess basic reproduction parameters of M. myotis from two nursery colonies over two gestation periods with different weather conditions. We found that ovulation probably occurred before female emergence from the hibernacula between late March and mid-April, triggered by a short period of high ambient temperature lasting at least three days. On the other hand, the estimated date of blastocyst implantation only varied within four days despite weather conditions differing significantly over the two years. Though specific foetal growth velocity did not differ between localities, the date of parturition was influenced by shelter temperature in May (difference between localities) and average ambient temperature in April (difference between years). The progesterone level does not appear to be a suitable indicator of pregnancy stage as it changed significantly during pregnancy and did not correlate with foetus size. The total gestation period (from fertilisation to parturition) varied between 56 and 73 days in our study.