The kingpin was last spotted by security cameras entering the shower area of the Altiplano prison, 90 kilometres west of Mexico City, on Saturday night before disappearing, the National Security Commission said.
An alarm was issued after "he was not visible" for a while and his cell was empty, the commission said in a statement in the early hours of today.
"An operation to locate him was deployed in the area and on roads of neighbouring states," it said, adding that flights were suspended at the nearby Toluca airport.
The Altiplano prison in central Mexico State houses the country's most notorious drug lords, murderers and kidnappers.
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The security commission did not have more details about the escape and a spokesman declined to comment, telling AFP a press conference was scheduled for today.
Guzman, 58, whose Sinaloa cartel shipped narcotics across the globe, was considered the world's most wanted drug lord before his arrest.
His first break from prison was in 2001, when he slipped past authorities by hiding in a laundry cart. He had been arrested in Guatemala in 1993.
Marines had recaptured him in February 2014 in a pre-dawn raid in a condo in Mazatlan, a Pacific resort in Sinaloa state, with the help of the US Drug Enforcement Administration.
His second escape is sure to embarrass the administration of President Enrique Pena Nieto, who was flying to France for a state visit when Guzman fled. The prison break will now certainly overshadow his trip.
Pena Nieto's government had won praise for capturing the powerful kingpin, a diminutive but feared man whose nickname means "Shorty."