We report the successful de novo sequencing of hemoglobin using a mass spectrometry-based approach combined with automatic data processing and manual validation for nine North American species with currently unsequenced genomes. The complete α and β chain of all nine mammalian hemoglobin samples used in this study were successfully sequenced. These sequences will be appended to the existing database containing all known hemoglobins to be used for identification of the mammalian host species that provided the last blood meal for the tick vector of Lyme disease, Ixodes scapularis.