Objectives This study evaluated the possible effects of ultrasound (US) on gene expression in brain tissue of rat embryos. Methods Four groups (n = 5 each) of pregnant Wistar Han rats were exposed to US for different durations (55, 100, 145, and 195 seconds) via a multifrequency transducer in the 2‐dimensional imaging mode with a pulse duration of 1.29 microseconds, a pulse repetition frequency of 1 kHz, and a derated spatial‐peak pulse‐average intensity of 222.4 W/cm2 on day 5, 9, 7, or 13 of gestation. Gene expression profiling was performed in fetal brain tissue (n = 5 per group) by quantitative reverse transcription–polymerase chain reaction arrays. Results The results indicated substantial alterations in gene expression. The most differentially expressed genes were Adamts5, Gadd45a, Npy2r, and Chrna1, which are implicated in important developmental signaling pathways. Conclusions On the basis of our findings, routine short US examinations for monitoring fetal development are not contraindicated, but prolonged exposures should be used only when needed to obtain important diagnostic information.