Ramon Cortines, superintendent of the Los Angeles Unified School District, is cooperating with the investigation, officials said.
Assistant Secretary for Civil Rights Russlynn Ali said only 3 percent of students in English Learner programs are proficient in math and English.
"At this time, we have reached no conclusion as to whether any violations of federal law exist," Ali said. "But the number of EL students and children of color in Los Angeles is large. It is critical that all students in the district receive equal access to a quality education. If civil rights violations are found, we will seek to put an end to them promptly."
About one-third of the district's students are in English Learner programs, the Los Angeles Times said.
The investigation is the department's first major formal civil rights action during the Obama administration, officials said.
"This is a big deal after eight years of lackluster enforcement," Thomas A. Saenz, president and general counsel of the Mexican American Legal Defense & Educational Fund, said.