As there was no maximum permissible limit prescription for aflatoxin B1 (AFB1) in Ethiopia, this study has been conducted to generate data on AFB1 levels in Ethiopian groundnut accessions/ landraces. Besides, an attempt was made to find out if there is any relationship between AFB1 and other parameters such as altitude of cultivation, individual seed weight, kernel colonization by Aspergillus flavus, total carbohydrates, protein and total free amino acids. Out of the 28 accessions studied, merely six accessions registered <= 2 ppb AFB1 and thus, they comply with maximum permissible limit set by European Union. Altitude of cultivation had no relationship with AFB1 levels. Interestingly, total carbohydrates in the seeds as well as kernel colonization by A. flavus showed statistically significant (p <0.01) positive relationships with AFB1 levels. It is suggested to use kernel colonization measurement as an alternative to the expensive ELISA based AFB1 measurement. Besides, suitable pre- and post-harvest aflatoxin management strategies should be developed to alleviate the AFB1 levels in Ethiopian groundnut.