The releases were part of a bargaining process in which Lukashenko is seeking a normalisation of ties with the US after years of isolation and sanctions, in exchange for easing repression in the former Soviet state he has led since 1994.
Belarusian human rights group Viasna said before Thursday's announcement there were more than 1100 political prisoners in the country.
One of those freed was Marfa Rabkova, the co-ordinator of Viasna's volunteer network, who was arrested in September 2020 and was serving a sentence of 14 years and nine months for "extremism" and other charges that she denied.
Supporters say she has suffered serious health problems in prison.
Bloggers, activists, journalists and protesters were also among those released after talks in the Belarusian capital Minsk between Lukashenko and John Coale, an envoy for US President Donald Trump.
Coale told Reuters he expected all remaining political prisoners to be released by the end of this year.
If this happened, the US would remove all sanctions placed on Belarus over the suppression of protests in 2020, he said.
Lukashenko was told on Thursday to cease arresting more political prisoners, Coale said.
"Hopefully, we made an impression on him that this is not okay behaviour," he said.
Lukashenko is an ally of Russian President Vladimir Putin and has supported Russia's invasion of Ukraine, although without sending Belarusian troops to fight there.
The US said it had agreed to drop sanctions on two Belarusian banks and the country's finance ministry but had made clear to Lukashenko that this must not facilitate other forms of sanctions evasion or support for Russia's war effort in Ukraine.
Coale said earlier that Lukashenko may soon visit the United States, a trip that would signal a breakthrough for the veteran authoritarian leader after years of being treated as a pariah because of human rights abuses and his backing for Putin in the war.
The US has signalled that it sees value in cultivating him, given his close ties with Putin, and says he has offered good advice on ending the four-year conflict.
Previous talks between Coale and Lukashenko resulted in the release of dozens of political prisoners last September and a further 123 in December, including Nobel Peace Prize winner Ales Bialiatski and opposition politicians Maria Kalesnikava and Viktar Babaryka.
The US responded then by removing sanctions on Belarusian potash - an ingredient in fertilisers and a major source of export revenue for the country of nine million people, which shares borders with Russia, Ukraine and three NATO members.
A US embassy spokesperson said 15 of those freed were being taken to Lithuania and the rest were staying in Belarus.
Many of those released in previous waves have been forced to leave the country immediately.
International Criminal Court prosecutors said on March 12 they were investigating these deportations as possible crimes against humanity.
"This was a pretty contentious negotiation over the last few months to keep the people in their home country," Coale told Reuters.
"These are people who had fairly low-level charges."
Rights activist Valiantsin Stefanovic, who stood trial alongside Bialiatski, was among those freed on Thursday.
Also released was Mikalai Kuliashou, a customs officer who had helped people cross the border into Lithuania in order to avoid arrest.
Exiled Belarusian opposition leader Sviatlana Tsikhanouskaya called the latest releases "a moment of great relief and hope," expressing gratitude to Trump and Coale.
"But we must be clear: many people are still behind bars," she said in a statement.
"Our goal remains unchanged — to free them all and to put a final end to repression."
