The importance of generation-old local knowledge in advancing agriculture is well recognized worldwide. However, such knowledge is continually eroding together with the extinction of locally evolved genetic materials. Consequently, the knowledge gap between different age groups is widening. The knowledge gap is also widening between male and female due to continuous shift in gender roles in agriculture. Using responses collected from 120 male and female farmers from Bara, the lowland Terai of Nepal, we assessed inter-generational and gender-based knowledge gap of smallholders on agricultural biodiversity by taking rice crop as an example. Based on the standard definition of younger and older generation, the age group was divided into two: ≤ 34 (15-34) years old and > 34 years old. Information was collected on: i) rice varieties recalled by respondents, ii) rice varieties recognized by observing standing crop on-farm, iii) rice varieties recognized by observing seed samples, and iv) respondent’s experiences and knowledge about selected cultivars. Descriptive analysis, t-test, and Pearson’s Correlation were used to analyze the data. Respondents of age group > 34 years old named and identified significantly (p < 0.01) more varieties than age group ≤ 34 in overall, indicating that the older generation is more knowledgeable and the knowledge hasn’t been adequately inherited to younger generations resulting in the erosion of knowledge. Similarly, males are significantly more knowledgeable than females in overall and among the higher age group category (p < 0.01), likely because males from Madhesi community have wider social networks both within and outside their villages than females. Females are more knowledgeable than males in the lower age group category because girls are engaged more in household chores and farming activities, while boys attain higher grades and travel for off-farm jobs. There was a strong correlation among the three techniques, namely, naming, recognizing standing crops, and recognizing the seeds (p < 0.01). This suggests that all the techniques are robust and can be interchangeably used for such type of studies depending on time and resource availability. This study finally infers that proper knowledge transfer is necessary to reduce the gulf of the knowledge gap between males and females as well as between generations if agrobiodiversity is to be conserved and utilized for growth and development of agriculture in the long run.