Paravertebral and intercostal nerve blocks have experienced a resurgence in popularity. Ultrasound has become the gold standard for visualization of the needle during injection of the analgesic, but the intercostal artery and vein can be difficult to visualize. We investigated the use of spectral analysis of raw radiofrequency (RF) ultrasound signals for identification of the intercostal vessels and six other tissue types in the intercostal and paravertebral spaces. Features derived from the one-dimensional spectrum, two-dimensional spectrum, and cepstrum were used to train four different machine learning algorithms. In addition, the use of the average normalized spectrum as the feature set was compared with the derived feature set. Compared to a support vector machine (SVM) (74.2%), an artificial neural network (ANN) (68.2%), and multinomial analysis (64.1%), a random forest (84.9%) resulted in the most accurate classification. The accuracy using a random forest trained with the first 15 principal components of the average normalized spectrum was 87.0%. These results demonstrate that using a machine learning algorithm with spectral analysis of raw RF ultrasound signals has the potential to provide tissue characterization in intercostal and paravertebral ultrasound.