The retailer has added the question to its annual Viewpoint survey, which quizzes staff on everything from whether they have the right equipment for their job, to how good they think their prospects for promotion are.
Some staff, commenting on an online forum, objected to the question. "I find this question a trifle intrusive, because it's a personal thing and absolutely nothing to do with Tesco," wrote one.
"Now what business is it of Tesco to know about what your sexuality is?" wrote another.
While it is common to ask for personal information such as age and ethnic origin, asking staff their sexuality is unusual. However, Tesco said that in five years time it may well be common practice.
"We are a responsible and inclusive retailer. We have a large and diverse workforce and it helps us better understand our staff and their needs," said a spokesman, adding that the questionnaire was anonymous and voluntary. "It may be the case that people decide not to respond and that is entirely their decision. If someone doesn't want to answer they don't have to."