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The Arsenal defender, 29, will be absent with a hamstring injury, despite having followed a rehabilitation programme that she hoped would enable her to play.
Sarina Wiegman's side can qualify for next year's World Cup in Brazil if they avoid defeat in Spain on Friday (20:00 BST).
They sit top of their group with four wins from four matches and have a three-point advantage over world champions Spain.
England take on Ukraine at Everton's Hill Dickinson Stadium on Tuesday, 9 June (20:00 BST).
Williamson, who led England to back-to-back European Championship victories in 2022 and 2025, has started just two Women's Super League games this season.
She had knee surgery following England's Euro 2025 victory and has played only three games for her country since.
Liverpool captain Grace Fisk has been called up to the squad to replace Williamson, while Brighton goalkeeper Sophie Baggaley also joins the Lionesses.
Baggaley will provide cover as Manchester City's Khiara Keating will miss the Spain game as she follows the return-to-play concussion protocols.
Keating missed Sunday's victory over Brighton in the FA Cup final, at which Baggaley was on the bench for the opposition.
A further assessment will determine whether Keating can be available for the Ukraine game, though regular number one Hannah Hampton is expected to start.
The squad arrive at St George's Park on Monday before travelling to Spain later in the week.
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