The 17-year-old Thompson has a one-stroke advantage over South Korea's Mi Jung Hur and Mindy Kim of the US with a 12-under 132 total.
"Playing at the highest level as a junior and amateur has helped me to play well at this point," said Thompson.
Hur and Kim each shot 65 and played in the same group on the links-style Senator course at the Robert Trent Jones Golf Trail's Capitol Hill complex.
Thompson matched the tournament record Thursday with a career-best 63.
Thompson was just 16 years, seven months and eight days old when she captured her lone win in eight tournaments in 2011, winning by eight strokes at the Navistar.
Her age record has since been eclipsed by 15-year-old New Zealander Lydia Ko, who claimed the Canadian Women's Open last month.
"I played pretty consistent, hit a few squirrely iron shots that I wasn't happy with, but overall I played consistent," said Thompson of her second round.
"My putting has gotten a lot better. It's all about confidence really and just getting up to it and saying to yourself you're going to make the putt."
Kim and Hur had opportunities to tie Thompson but Hur missed a three-foot birdie putt on the par-five eighth, their 17th hole of the day, and Kim missed a six-footer on the par-four ninth.
Dori Carter was two strokes back at 10-under, along with Hee Young Park and Lizette Salas. Carter had a 67, and Park and Salas shot 69.
Top-ranked Yani Tseng of Taiwan was three-under after a 70. Michelle Wie missed the cut by a stroke, shooting a 73 to finish at even par. – AFP