This paper investigates the possibility of using spatial coding based on frequency division for in-vivo synthetic transmit aperture (STA) ultrasound imaging. When using spatial encoding for STA, it is possible to use several transmitters simultaneously and separate the signals at the receiver. This increases the maximum transmit power compared to conventional STA, where only one transmitter can be active. The signal-to-noise-ratio can therefore be increased and better penetration can be obtained. For frequency division, the coding is achieved by designing a number of transmit waveforms with disjoint spectral support, spanning the passband of the ultrasound transducer. The signals can therefore be separated at the receiver using matched filtering. The method is tested using a commercial linear array transducer with a center frequency of 9 MHz and 68% fractional bandwidth. In this paper, the transmit waveforms are designed as non-linear frequency modulated signals. This allows for efficient design of the amplitude spectrum of the signals. The duration of the signals was 25 μs and the bandwidth of each frequency band was 2.8 MHz. Eight frequency bands were designed which allowed for four transmitters to be active simultaneously. The method is compared to traditional STA with linear frequency modulation as means of temporal coding. The reference waveform was a 20 μs chirp at 9.37 MHz with a bandwidth of 11.3 MHz. Penetration and resolution is evaluated using a tissue mimicking phantom. The increase in penetration for the frequency division method was approximately 2 cm. The SNR was measured in the same type of phantom and an increase in SNR at depths between 3 cm and 10 cm of 7.2±3.6 dB was found. In-vivo experiments were carried out by an experienced sonographer. First, the common carotid artery was scanned on a 27 year old healthy male volunteer. The image quality was comparable for the two methods. To compare penetration depth of the two methods, the vesica fellea was scanned on the same volunteer. The frequency division method exhibited approximately 2 cm improvement in penetration compared to conventional STA.