Zapatista officials in a village outside the township of Chilon said paramilitary gunmen affiliated with a radical right-wing group known as "Los Aguilares" gunned down Zapatista leader Antonio Mejia.
In a statement, the Zapatistas claimed the paramilitary group was funded by members of the Institutional Revolutionary Party(PRI), the faction that ruled Mexico from 1929-2000 and still controls Chilon's government.
In a statement, local PRI officials said they had nothing to do with the killing.
Authorities in Chilon, 90 miles (140 kilometers) north of the mountain city of San Cristobal de Las Casas, said they were investigating the killing and had not yet determined a motive.
The Zapatistas staged a 12-day rebellion in the name of Indian rights in Jan.1994. They have been officially at war with the government ever since, though they have remained quiet for more than a year.
Also on Sunday, two other Zapatista leaders were shot and killed during a meeting of Zapatista leadership outside the Chiapas city of Ocosingo, 25 miles (40 kilometers) from Chilon.
Since the 1994 rebellion, in Ocosingo, Chilon and villages across Chiapas, Zapatista supporters have chosen a local rebel governing board that performs city duties alongside the official government.
Local authorities insist the attack that claimed two victims had nothing to do with politics and instead stemmed from a disagreement between two local families.
But Zapatista leaders say the gunmen were loyal to the PRI which control Ocosingo's government and has had several high-profile disagreements with Zapatista leaders in recent months. Party officials have denied those charges.