Exome and genome sequencing (ES/GS) are routinely used for the diagnosis of genetic diseases in developed countries. However, their implementation is limited in countries from Latin America. We aimed to describe the results of GS in patients with suspected rare genetic diseases in Colombia. We studied 501 patients from 22 healthcare sites from January to December 2022. GS was performed in the index cases using dried blood spots on filtercards. Ancestry analysis was performed under iAdmix. Multiomic testing was performed when needed (biomarker, enzymatic activity, RNA-seq). All tests were performed at an accredited genetic laboratory. Ethnicity prediction data confirmed that 401 patients (80%) were mainly of Amerindian origin. A genetic diagnosis was established for 142 patients with a 28.3% diagnostic yield. The highest diagnostic yield was achieved for pathologies with a metabolic component and syndromic disorders (p < 0.001). Young children had a median of 1 year of diagnostic odyssey, while the median time for adults was significantly longer (15 years). Patients with genetic syndromes have spent more than 75% of their life without a diagnosis, while for patients with neurologic and neuromuscular diseases, the time of the diagnostic odyssey tended to decrease with age. Previous testing, specifically karyotyping or chromosomal microarray were significantly associated with a longer time to reach a definitive diagnosis (p < 0.01). Furthermore, one out of five patients that had an ES before could be diagnosed by GS. The Colombian genome project is the first Latin American study reporting the experience of systematic use of diagnostic GS in rare diseases.