Yaya Dillo Djerou was the leading opponent and a cousin of Mahamat Idriss Deby Itno, who was proclaimed transitional president by the country's ruling junta in 2021.
Dillo, 49, died on Wednesday after troops surrounded the office of his Socialist Party Without Borders in the capital N'Djamena, in what the party says was an "execution."
The government denied the accusation, saying there was an exchange of gunfire when Dillo "opposed his arrest."
"The killing of a potential presidential candidate during an assault by Chadian security forces on an opposition party headquarters raises serious concerns about the environment for elections scheduled for May 6," HRW said in a statement.
"The circumstances of Yaya Dillo's killing are unclear, but his violent death highlights the dangers facing opposition politicians in Chad, particularly as elections approach," said Lewis Mudge, Central Africa director at HRW.
"The lack of clarity surrounding the attack on the PSF headquarters, the threats previously faced by Dillo, and the general political repression in the country all point to the need for an independent investigation with foreign assistance into the February 28, 2024 events," HRW said.
The rights organisation added that it had "reviewed several photos sent by a reliable source close to Dillo, showing him dead with a single bullet wound to his head."
The violence came a day after Chad's military rulers announced a presidential election for May 6.
The voting would end a three-year transition period and aim to restore constitutional rule.
Dillo had told AFP before his death that people wanted to "physically eliminate me" ahead of the election in which he — and Deby Itno — planned to run.
The government had accused Dillo of leading an attack against the offices of Chad's internal security agency the night before his death. He denied any involvement.
Deby Itno took power after the death of his father, veteran leader Idriss Deby Itno, in 2021 while fighting rebels.
He promised a return to civilian rule and elections within 18 months, but extended the transition by two years.
Human Rights Watch Urges Investigation of Chadian Opposition Figure
FILE —Chad former presidential election candidate Yaya Dillo gives a press conference on April 30, 2021 in N'djamena.
LIBREVILLE —Human Rights Watch on Saturday called for a foreign-backed independent investigation into the killing of Chad's leading opposition figure ahead of a long-awaited election.