Abstract: Subsistence Bhutanese farmers spread across different agro-ecological zones maintain large species and varietal diversity of different crops in their farm. However, no studies have been undertaken yet to assess why farmers conserve and maintain large agro-biodiversity, the extent of agro-ecological richness, species richness, estimated loss of traditional varieties and threats to the loss of on-farm agro-biodiversity. Information on the number of varieties cultivated by the farmers for six important staple crops were collected from nine districts and twenty sub-districts spread across six different agro-ecological zones of the country to understand farmers reasons for maintaining on-farm crop diversity, estimate agro-ecological richness, species richness and the overall loss of traditional varieties, to know the famers’ level of awareness on climate change and the different threats to crop diversity. The results from this study indicated that an overwhelming 93% of the respondents manage and use agro-biodiversity for household food security and livelihood. The average agro-ecological richness ranged from 1.17 to 2.26 while the average species richness ranged from 0.50 to 2.66. The average agro-ecological richness indicates a large agro-ecological heterogeneity in terms of the different species of staple crops cultivated. The average species richness on the other hand shows that agro-ecological heterogeneity determines the type and extent of the cultivation of the six different staple cereals under consideration. The overall loss of traditional varieties in a time period of 20 years stands at 28.57%. On climate change, 94% of the farmers recognize that local climate is changing while 86% responded that they are aware of the potential impacts of climate change on their livelihoods. Climate change and associated factors was considered the most imminent threat to the management and loss of on-farm agro-biodiversity. The results from this study indicate that on-farm agro-biodiversity conservation, development and utilization programs have to be more specific to the different agro-ecological zones considering the agro-ecological heterogeneity. Attention has to be given to individual crops that have low average species richness and high percentage of loss of traditional varieties. The impact of climate change could offset the traditional seed system which primarily supports the persistence of on-farm agro-biodiversity in several ways.