Williams will miss the WTA Premier Mandatory event played on clay courts in the Spanish capital, starting Sunday.
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Injured Venus Williams out of Madrid Open
Former world number one Venus Williams has pulled out of the Madrid Open due to a right hamstring injury.
The injury is a blow to the 35-year-old's preparations for the French Open, with the main draw of the year's second grand slam starting on May 22.
Williams, a seven-time major winner, had found some form with a title win in Kaohsiung earlier this year.
However, she made early exits at Indian Wells, Miami and Charleston.
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