Selenium (Se) is an essential micronutrient for human and animal health. Globally, more than one billion people are Se deficient due to low dietary Se. Low dietary intake of Se can be improved by Se supplementation, food fortification and biofortification of crops. Lentil (Lens culinaris Medikus subsp. culinaris) is a popular cool-season food legume in many parts of the world; it is naturally rich in Se and therefore has potential for Se biofortification. An Se foliar application experiment at two locations and a multi-location trial of 12 genotypes at seven locations were conducted from April to December 2011 in South Australia and Victoria, Australia. Foliar application of a total of 40 g/ha of Se as potassium selenate (K2SeO4) – 10 g/ha during full bloom and 30 g/ha during the flat pod stage – increased seed Se concentration from 201 to 2772 μg/kg, but had no effect on seed size or seed yield. Consumption of 20 g of biofortified lentil can supply all of the recommended daily allowance of Se. After Se foliar application, cultivars PBA Herald XT (3327 μg/kg), PBA bolt (3212) and PBA Ace (2957 μg/kg) had high seed Se concentrations. These cultivars may be used in lentil biofortification. In the genotypic evaluation trial, significant genotype and location variation was observed for seed Se concentration, but the interaction was not significant. In conclusion, foliar application of Se as K2SeO4 is an efficient agronomic approach to improve seed Se concentration for lentil consumers and there is also scope for genetic biofortification in lentil.